Pharmaceutical compositions, methods, and kits for treatment and diagnosis of lung cancer

ABSTRACT

The present invention relates to pharmaceutical compositions, methods and kits that provide for the early diagnosis and treatment of lung cancer. More particularly, the present invention relates to pharmaceutical compositions containing uteroglobin for preventing or inhibiting metastasis of lung tumor cells and methods of using the same to prevent or inhibit metastasis of lung tumor cells. The present invention also relates to methods and kits for early diagnosis of metastatic lung cancer by assaying for uteroglobin and comparing the results against control cells. The present invention also relates to methods and kits for detection of metastatic lung cancer by assaying for the presence of an aberrant form of uteroglobin.

[0001] (This application is a continuation-in-part of U.S. patentapplication Ser. No. 08/658,796, filed Jun. 5, 1996, which is acontinuation-in-part of U.S. patent application Ser. No. 08/486,203,filed Jun. 7, 1995, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/400,084, filed on Mar. 7, 1995, the entirety ofeach are incorporated by reference herein.)

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to pharmaceutical compositions,methods and kits that provide for the early diagnosis and treatment oflung cancer. More particularly, the present invention relates topharmaceutical compositions containing uteroglobin for treating orinhibiting metastasis of lung epithelial tumor cells and methods ofusing the same to treat or inhibit metastasis of lung epithelial tumorcells. The present invention also relates to methods and kits for earlydiagnosis of metastatic lung cancer by assaying for uteroglobin andcomparing the results against control cells.

[0004] 2. Description of the Prior Art

[0005] Cancers develop from uncontrolled multiplication of cells. Allcancers are life threatening, and lung cancer remains the major cause ofcancer death among both males and females.

[0006] There are four types of lung cancer found in humans: squamous,adeno, small cell, and large cell. Each tumor expresses specificdifferentiation features or surface phenotype determinants, all of whichdistinguish these cells form normal cells. The development of monoclonalantibody diagnostic techniques has greatly enhanced the production ofreagents capable of differentiating normal cells from cancer cells anddifferentiating between cancer cell types. However, none of thesemarkers have been able to provide information concerning when a tumorcell or cells will become metastatic.

[0007] The major cause of morbidity and mortality for cancer in humansis metastatic disease. As a consequence, there has been much interest inthe mechanisms involved in invasion of cells and metastasis. Severalenzyme systems have been implicated in the metastatic process:metalloproteinases, cysteine proteases, and serine proteases. Yagel, S.A. et al., 49 Cancer Research 3553 (1989), Dickson, R. B., 41 J. SteroidBiochem. Molec. Biol. 389 (1992) and Zucker S. et al., 45 CancerResearch 6168 (1985). Inhibitors of metalloproteinases, especially ofthe collagenases, have been the focus of intense study. DeClerck A. etal., 52 Cancer Research 701 (1992).

[0008] However, metastatic cancers have proven to be particularlydifficult to treat. These cancers pose the highest risk to patients and,for optimal prognosis, often must be treated by aggressive methods thatpresent increased risks of deleterious side-effects. Most treatments nowavailable have severe toxic side effects to the human body such asnausea, vomiting, hair loss and fatigue.

[0009] Therefore, there is a great need for methods that accuratelydistinguish those tumors that are likely to metastasize from those thatare unlikely to do so. Furthermore, since currently available methods oftreating metastatic cancers often are inadequate, there also is a clearneed for improved anti-metastatic agents and methods to treat metastaticcancers, and metastatic lung cancers in particular.

[0010] Metastatic cancers originate from a primary tumor. Metastasis ofthe primary tumor produces secondary tumors and disseminated cancer. Itis well known that both primary and secondary tumors shed large numbersof cells.

[0011] The shed cells can spread through the body. For instance, aprimary tumor may damage the surrounding lymph or circulatory vessels,allowing entry of shed cells into the lymph or circulatory systems, andhastening their spread in the body. Moreover, shedding of cells bycancerous tumors increases during surgery and radiotherapy.

[0012] Most shed cells do not form new tumors. To do so such cells mustsurmount a series of physical and physiological barriers. In fact, aseries of distinct events must occur for metastasis to occur.

[0013] The primary tumor physically must (i) invade interstitial spaceof the primary tissue. In particular, it must (ii) penetrate thebasement membrane of the tissue. For most metastases, the tumor mustdamage the endothelial cell wall of lymphatic or vascular vessels toprovide access to shed cells. Cells that enter the lymph or blood must(iii) survive hemodynamic stress and host defenses in the circulationand, furthermore, (iv) the cells must lodge at a new site in thecirculatory system, a process that apparently involves aggregatedplatelets. A cell then must (v) extravasate out of the vessel into theinterstitial space. Finally, it must (vi) invade the interstitial spaceof the secondary organ and proliferate in the new location. Although theprocess of metastasis is physiologically complex, the overall pattern ofmetastasis is general to many types of cancers including lung cancers.

[0014] The metastatic process also clearly involves complexintracellular mechanisms that alter cancerous cells and theirinteractions with surrounding cells and tissues. Currently, it isthought that proliferation of many cancerous cells depends upon specificligand-receptor interactions. Thus far, however, it has not beenpossible to develop a therapy that prevents or effectively inhibitsmetastasis of metastatic cancers.

[0015] The complexity of the processes involved in metastasis, and thelack of understanding of underlying molecular mechanisms, have made itparticularly difficult in some cases to distinguish tumors that arelikely to metastasize from those that are unlikely to do so.

[0016] The inability to discern the metastatic potential of tumorsprecludes accurate prognosis and leads, inevitably, to the therapeuticintervention that either is too aggressive or insufficiently aggressive.Furthermore, for all types of cancers it has been difficult orimpossible thus far to develop treatments that inhibit or prevent thespread of metastatic tumors. Clearly, there remains a great need formethods to accurately determine the metastatic potential of tumors andfor effective anti-metastatic compositions and methods.

[0017] The development of monoclonal antibody techniques fordifferentiating normal cells from cancer cells and differentiating onetype of cancer cell from another has greatly enhanced lung cancerdetection but does not yield information regarding metastatic potentialof a neoplastic, dysplastic, or tumor cell.

[0018] Mulshine et al., U.S. Pat. No. 5,455,159, discloses earlydiagnosis of lung cancer by using monoclonal antibodies to detect cellsthat express antigens whose increased levels correlates with thedevelopment of lung cancer.

[0019] Mulshine et al., U.S. Pat. No. 4,569,788, discloses monoclonalantibodies which can be used to detect non-small cell lung cancer anddistinguish non-small cell cancer from other cancers and normal cells.

[0020] Hirohashi et al., U.S. Pat. No. 4,683,200, discloses IgM classmonoclonal antibody which is reactive with human lung cancers.

[0021] Loor et al., U.S. Pat. No. 4,690,890, discloses a process fordetecting two antigens using an immunometric dual sandwich assay.

[0022] Tanswell et al., U.S. Pat. No. 4,624,930, discloses a process fordetecting polyvalent antigens using a three receptor reaction.

[0023] However, none of the prior art techniques utilize uteroglobin orantibodies which react with the same. None of the prior art techniquesdisclose the use of proteins which are down-regulated or which exhibitdecreased expression of the normal protein during neoplasticdevelopment. In contrast, the prior art has generally relied upon theup-regulation of cellular proteins to support the detection of thesetumor markers. Further, the prior art does not disclose aberrantlyprocessed proteins leading to aberrant protein structure and function,nor does the prior art provide for the ability to inhibit the metastaticprocess by administering the missing normal protein, thus re-creatingpositive feedback mechanisms within the cellular machinery.

SUMMARY OF THE INVENTION

[0024] The present invention relates to pharmaceutical compositions,methods and kits that provide for the early diagnosis and treatment oflung cancer. More particularly, the present invention relates topharmaceutical compositions containing uteroglobin for treating orinhibiting metastasis of lung epithelial tumor cells and methods ofusing the same to treat or inhibit metastasis of lung epithelial tumorcells. The present invention also relates to methods and kits for earlydiagnosis of metastatic lung cancer by assaying for uteroglobin andcomparing the results against control cells.

[0025] Accordingly, the present invention provides a method fortreatment of metastatic lung cancer, comprising administering atherapeutically effective amount of an inhibitor of phospholipase A₂ toan animal.

[0026] The present invention also provides a pharmaceutical compositionfor treatment of metastatic lung cancer in an animal, comprising atherapeutically effective amount of an inhibitor of phospholipase A₂ anda pharmaceutically acceptable carrier.

[0027] The present invention is also directed to a method for inhibitingmetastasis of lung epithelial tumor cells, comprising administering atherapeutically effective amount of an inhibitor of phospholipase A₂ toan animal.

[0028] The present invention is further directed to a pharmaceuticalcomposition for inhibiting metastasis of lung epithelial tumor cells inan animal, comprising a therapeutically effective amount of an inhibitorof phospholipase A₂ and a pharmaceutically acceptable carrier.

[0029] The present invention additionally provides for a method forinhibiting invasion of lung epithelial tumor cells, comprisingadministering a therapeutically effective amount of an inhibitor ofphospholipase A₂ to an animal.

[0030] A pharmaceutical composition for inhibiting invasion of lungepithelial tumor cells in an animal is also provided and comprises atherapeutically effective amount of an inhibitor of phospholipase A₂ anda pharmaceutically acceptable carrier.

[0031] In addition to methods and compositions for treating metastaticlung cancer cells, the present invention also relates to diagnosticmethods and kits.

[0032] One preferred method is directed to a method for detecting oridentifying metastatic lung cancer, comprising: comparing an amount ofan inhibitor of phospholipase A₂ in a sample of lung epithelial tissueto at least one reference which correlates to the amount of inhibitor ofphospholipase A₂ in normal lung epithelial tissue, or to metastatic lungcancer, whereby differential amounts of the inhibitor of phospholipaseA₂ between the sample of lung epithelial tissue and the reference,detects or identifies metastatic lung cancer.

[0033] Another preferred method includes detecting or identifyingmetastatic lung cancer, comprising: assaying for inhibitor ofphospholipase A₂ in a sample of lung epithelial tissue; and comparingthe amount of the inhibitor in the sample to a reference whichcorrelates to the amount of the inhibitor in normal lung epithelialcells or metastatic lung epithelial cells, wherein differential amountsof the inhibitor of between the sample and the reference detects oridentifies metastatic lung cancer.

[0034] An additional preferred method contemplates detecting oridentifying a pathological condition of lung epithelial tissue,comprising: assaying for inhibitor of phospholipase A₂ in a sample oflung epithelial tissue; and comparing the amount of the inhibitor in thesample to a reference which correlates to the amount of the inhibitor innormal lung epithelial tissue or metastatic lung epithelial cells,wherein differential amounts of the inhibitor between the sample and thereference detects or identifies a pathological condition of lung tissue.

[0035] A further preferred embodiment of the present invention includesa method for detecting or identifying an inhibitor of phospholipase A₂in a sample of lung epithelial tissue, comprising: assaying for aninhibitor of phospholipase A₂ in a sample of sample of lung epithelial.

[0036] Another particularly preferred embodiment of the inventionrelates to the detection of aberrant uteroglobin protein. Surprisingly,an aberrant form of native uteroglobin is expressed and directlycorrelates to the decrease in native or normal uteroglobin as metastasisprogresses. Accordingly, one aspect of the present invention involvesnot only the detection of the loss of normal uteroglobin as a gauge ofmetastatic or invasive activity, but also the increase in the level ofaberrant uteroglobin. This increase provides a positive detectionwhereas the loss of normal uteroglobin relies upon the absence.Furthermore, this aberrant form of uteroglobin is secreted in sputum andbronchial fluid, as well as other related bodily fluids such asrespiratory fluids and the like where such proteins are found.Accordingly, a method and kit for detection of an aberrant form ofuteroglobin as a direct indicator of metastasis and invasiveness isprovided.

[0037] Preferred kits of the present invention include a kit fortreatment of lung cancer, comprising a therapeutically effective amountof an inhibitor of phospholipase A₂ in a pharmaceutically acceptablecarrier and a device for delivery of the inhibitor to the lung cancer,wherein the inhibitor, the carrier and the device are packaged in acontainer.

[0038] An additional preferred kit contemplates inhibiting metastasis oflung epithelial tumor cells, comprising a therapeutically effectiveamount of an inhibitor of phospholipase A₂ in a pharmaceuticallyacceptable carrier and a device for delivery of the inhibitor to thelung epithelial tumor cells, wherein the inhibitor, the carrier and thedevice are packaged in a container.

[0039] A further kit of the present invention is a kit for inhibitinginvasion of lung epithelial tumor cells, comprising a therapeuticallyeffective amount of an inhibitor of phospholipase A₂ in apharmaceutically acceptable carrier and a device for delivery of theinhibitor to the lung epithelial tumor cells, wherein the inhibitor, thecarrier and the device are packaged in a container.

[0040] The present invention also provides a kit for detecting oridentifying metastatic lung cancer, comprising: a) means for collectinga sample of lung epithelial cells; b) means for detecting an inhibitorof phospholipase A₂ in the sample of lung epithelial cells; and c) atleast one reference which correlates to the amount of inhibitor ofphospholipase A₂ in normal lung epithelial cells or in metastatic lungcancer cells, wherein differential amounts of the inhibitor ofphospholipase A₂ between the sample and the reference detects oridentifies metastatic lung cancer.

[0041] Another preferred kit is a kit for detecting or identifying apathological condition of lung epithelial cells, comprising: a) meansfor collecting a sample of lung epithelial cells; b) means for detectingan inhibitor of phospholipase A₂ in the sample of lung epithelial cells;and c) at least one reference which correlates to the amount ofinhibitor of phospholipase A₂ in normal lung epithelial cells or inmetastatic lung cancer cells, wherein differential amounts of theinhibitor of phospholipase A₂ between the sample and the referencedetects or identifies a pathological condition of the lung epithelialcells.

[0042] A further kit of the present invention involves a kit fordetecting or identifying an inhibitor of phospholipase A₂ in a sample oflung epithelial cells, comprising: a) means for collecting a sample oflung epithelial cells; and b) means for detecting an inhibitor ofphospholipase A₂ in the sample of lung epithelial cells.

BRIEF DESCRIPTION OF THE FIGURES

[0043]FIG. 1 is a bar chart showing the dose effect of uteroglobin onthe invasiveness of cells of the TSU-Pr1 and DU-145 cell lines,epithelial cell lines which correlate to lung epithelium but which arederived from human prostate tumors. Both TSU-Pr1 and DU-145 cells areandrogen independent. Cells were cultured in zero, 0.01, 0.1 and 1.0 μMuteroglobin for 24 hr. Invasiveness then was assayed by migrationthrough filters coated with reconstituted basement membrane (RBM) inresponse to serum free or fibroblast conditioned media (FCM). Invadingcells were stained with crystal violet, the dye was extracted andinvasiveness was determined by measuring dye concentration by opticalabsorbance at 585 nm. Each point in the graph is the mean of results ofthree separate experiments, each carried out using triplicate cultures.The bars show the standard error of the mean for each point.

[0044]FIG. 2 is a bar chart showing the dose effect of uteroglobin onthe invasiveness of cells of the PC3-M and LNCaP cell lines, epithelialcell lines which correlate to lung epithelium but which are derived fromhuman prostate tumors. LNCaP cells are androgen-sensitive. PC3-M cellsare androgen independent. Assays were performed as described in thecaption to FIG. 1.

[0045]FIG. 3 is a bar chart showing that myoglobin, albumin andheat-inactivated uteroglobin do not affect the invasiveness of DU-145cells. Assays were performed essentially as described in the caption toFIG. 1.

[0046]FIG. 4 is a graph showing a time course of the effect ofuteroglobin on invasiveness of FCM-stimulated DU-145 cells. Cells wereincubated without or with 1.0 μM uteroglobin for 3, 6, 12, or 24 hr. andthen assayed for invasion in response to FCM as described in the captionto FIG. 1.

[0047]FIG. 5 is a graph showing that uteroglobin inhibits arachidonicacid release from FCM-stimulated DU-145 cells over a five hour period.Cells were incubated for 24 hr. in αMEM/SF media containing ¹⁴C-labeledarachidonic acid. Free label then was washed away, and the cells wereincubated in FCM without and with 1 μM uteroglobin. Arachidonic acidrelease was measured by ¹⁴C released from the cells into the medium.

GLOSSARY

[0048] Arachidonic Acid Cascade: a series of enzymatic reactions thatresults in the production and release of arachidonic acid by a cell. Thecascade is sensitive to ligand signals and arachidonic acid itself is anautocrine factor.

[0049] Control: a reference against which a test outcome is compared togauge results. A series of standards is a plurality of such referencesthat represent points along a qualitative or a quantitative scale.

[0050] Detecting: includes assaying, imaging, or otherwise establishingthe presence or absence of the target protein, target cell, nucleicacid, subunits thereof, or combinations of reagent bound targets, andthe like.

[0051] DU-145: an epithelial cell line derived from a human prostatetumor, which is androgen independent.

[0052] Effector: a substance that inhibits, modifies, engenders, alters,modulates or controls an activity of a cell; a substance that caninhibit, modify, engender, alter, modulate or control a physiologicalactivity of a cell or organism. Typically, a protein, such as an enzyme,cofactor or transcription regulatory protein, or an activator orinhibitor of an enzyme, an enzyme complex, a receptor or a receptorcomplex, for instance.

[0053] Epithelial Cell Origin: derived from an epithelial cell, ofwhatever tissue.

[0054] FCM: fibroblast conditioned media

[0055] Identifying: assaying for, imaging, ascertaining, establishing,or otherwise determining one or more factual characteristics of lungneoplasia, dysplasia, lung cancer, lung epithelial tumor cells,metastasis, or a similar conditions.

[0056] Inhibiting/Inhibition: The term “inhibiting” or “inhibition”, inthe context of tumor growth or tumor cell growth, may be assessed bydelayed appearance of primary or secondary tumors, slowed development ofprimary or secondary tumors, decreased occurrence of primary orsecondary tumors, slowed or decreased severity of secondary effects ofdisease, arrested tumor growth and regression of tumors, among others.In the extreme, complete inhibition, is referred to herein asprevention.

[0057] Inhibition: inhibition of metastasis or invasion may be measuredby many parameters in accordance with the present invention and, forinstance, may be assessed by delayed appearance of secondary tumors,slowed development of primary or secondary tumors, decreased occurrenceof secondary tumors, slowed or decreased severity of secondary effectsof disease, arrested tumor growth and regression of tumors, failure oftumor cells to extravasate, intravasate, or otherwise migrate throughthe basement membrane, and failure of tumor cells to penetrateepithelial basement membrane. In the extreme, complete inhibition, isreferred to herein as prevention.

[0058] The term “inhibition”, in the context of enzyme inhibition,relates to reversible enzyme inhibition such as competitive,uncompetitive, and noncompetitive inhibition. This can be experimentallydistinguished by the effects of the inhibitor on the reaction kineticsof the enzyme, which may be analyzed in terms of the basicMichaelis-Menten rate equation. Competitive inhibition occurs when theinhibitor can combine with the free enzyme in such a way that itcompetes with the normal substrate for binding at the active site. Acompetitive inhibitor reacts reversibly with the enzyme to form anenzyme-inhibitor complex [EI], analogous to the enzyme-substratecomplex:

E+I==EI

[0059] Following the Michaelis-Menten formalism, we can define theinhibitor constant, K_(i), as the dissociation constant of theenzyme-inhibitor complex:$K_{i} = \frac{\lbrack E\rbrack \lbrack I\rbrack}{\left\lbrack {E\quad I} \right\rbrack}$

[0060] Thus, in accordance with the above and as used herein, K_(i) isessentially a measurement of affinity between a molecule, and itsreceptor, or in relation to the present invention, between the presentinventive compounds and the enzyme to be inhibited. It should be notedthat IC50 is a related term used when defining the concentration oramount of a compound which is required to cause a 50% inhibition of thetarget enzyme.

[0061] Lung Cancer: includes benign and metastatic carcinomas,adenocarcinomas, epithelial cell tumors, neoplasia, dysplasia, andhyperplasia of lung and lung associated tissues such as bronchialepithelium, pleural tissues, alveolar tissues. Lung Cancer may becharacterized by a high nuclear/cytoplasmic ratio, hyperchromasia,coarsely granular chromatin, absence of nucleoli, isolated cells andcellular and nuclear pleomorphism.

[0062] LNCaP: an illustrative and correlative epithelial cell linederived from a human prostate tumor, which is androgen sensitive. TheLNCaP cell line was derived from a supraclavicular lymph node metastasisof a human prostate carcinoma. Cells of this line exhibit increasedproliferation in response to androgen, in vitro, and they secreteprostate specific antigen (PSA), a marker of differentiated epithelialcells. As used herein, they show the correlation between the ability ofuteroglobin and inhibitors of PLA₂ to inhibit growth of epithelial cellderived tumors, including lung epithelial tumors.

[0063] Metastasis: As set out in Hill, R. P., Chapter 11, Metastasis,pp178-195 in The Basic Science of Oncology, Tannock et al., Eds.,McGraw-Hill, New York (1992), which is incorporated by reference hereinin its entirety, metastasis is “The ability of cells of a cancer todisseminate and form new foci of growth at noncontiguous sites (i.e., toform metastases).”

[0064] Similarly, metastasis is described in Aznavoorian et al., Cancer71: 1368-1383 (1993), which is incorporated by reference herein in itsentirety, as “The transition from in situ tumor growth to metastaticdisease is defined by the ability of tumor cells of the primary site toinvade local tissues and to cross tissue barriers. . . . To initiate themetastatic process, carcinoma cells must first penetrate the epithelialbasement membrane and then invade the interstitial stroma. . . . Fordistant metastases, intravasation requires tumor cell invasion of thesubendothelial basement membrane that must also be negotiated duringtumor cell extravasation . . . The development of malignancy is alsoassociated with tumor-induced angiogenesis [which] not only allows forexpansion of the primary tumor, but also permits easy access to thevascular compartment due to defects in the basement membranes of newlyformed vessels.”

[0065] Mimetic: a molecule which, in shape and effect, mimics the shapeand therefore the activity of another molecule or complex of moleculesupon which it is designed.

[0066] Mutein: An amino acid sequence variant of a protein. Thevariation in primary structure may include deletions, additions andsubstitutions. The substitutions may be conservative ornon-conservative. The differences between the natural protein and themutein generally conserve desired properties, mitigate or eliminateundesired properties and add desired or new properties. In the presentinvention the muteins generally are those that maintain or increaseanti-metastatic activity. Particularly, uteroglobin muteins are aminoacid sequence variants of uteroglobin that maintain or increase theanti-metastatic activity of uteroglobin.

[0067] NSAID: Nonsteroidal anti-inflammatory agents. Small moleculedrugs, as the term is used herein, that inhibit cyclooxygenase, but donot directly inhibit phospholipase A₂. These compounds have been usedfor their anti-inflammatory action. Aspirin, phenylbutazone, ibuprofen,sulfinpyrazone (Anturane) and indomethacin are NSAIDs.

[0068] Pathological Condition: The term “pathological condition” relatesto lung cancers and related diseases such as epithelial cell tumors,metastatic carcinomas, adenocarcinomas, conditions characterized byabnormal growth of lung epithelial cells, bronchial cells, pleuraltissues, and other conditions requiring treatment by the inhibitors ofthe present invention.

[0069] PC3-M: an illustrative and correlative epithelial cell linederived from a human prostate tumor, which is androgen independent. Asused herein, they show the correlation between the ability ofuteroglobin and inhibitors of PLA₂ to inhibit growth of epithelial cellderived tumors, including lung epithelial tumors.

[0070] Peptide Analog: an oligo or polypeptide having an amino acidsequence of or related to a protein. Peptide analogs of the presentinvention are peptides that have anti-metastatic activity and an aminoacid sequence the same as or similar to a region of an anti-metastaticprotein, such as uteroglobin.

[0071] PLA: phospholipase A

[0072] PLA₂: phospholipase A₂

[0073] Prevention: The term “prevention”, in relation to tumor growth ortumor cell growth, means no tumor or tumor cell growth if none hadoccurred, no further tumor or tumor cell growth if there had alreadybeen growth.

[0074] In relation to metastasis, virtually complete inhibition, nometastasis if it had not occurred, no further metastasis if there hadalready been metastasis of a cancer. See INHIBITION.

[0075] PSA: prostate specific antigen

[0076] RBM: reconstituted basement membrane. A multicomponent matrixapproximating the molecular composition of the intracellular tissuematrix and the epithelial cell basement membrane. Preparations forpreparing RBM are well known and are available from commercialsuppliers.

[0077] SFM: Serum free medium

[0078] Treatment: The term “treatment” refers to any process, action,application, therapy, or the like, wherein an animal, including a humanbeing, is subject to medical aid with the object of improving theanimal's condition, directly or indirectly.

[0079] TSU-Pr1: an illustrative and correlative epithelial cell linederived from a human prostate tumor, which is androgen independent. Asused herein, they show the correlation between the ability ofuteroglobin and inhibitors of PLA₂ to inhibit growth of epithelial cellderived tumors, including lung epithelial tumors.

[0080] UG: uteroglobin

[0081] hUG: human uteroglobin

DETAILED DESCRIPTION OF THE INVENTION

[0082] The present invention relates to pharmaceutical compositions,methods and kits that provide for the early diagnosis and treatment oflung cancer. More particularly, the present invention relates topharmaceutical compositions containing uteroglobin for treating orinhibiting metastasis of lung epithelial tumor cells and methods ofusing the same to treat or inhibit metastasis of lung epithelial tumorcells. The present invention also relates to methods and kits for earlydiagnosis of metastatic lung cancer by assaying for uteroglobin andcomparing the results against control cells.

[0083] DETECTING OR IDENTIFYING METASTATIC TUMORS

[0084] Assaying for the absence of inhibitors of PLA₂ such asuteroglobin in cells directly correlates to whether or not a tumor maymetastasize. Detecting the metastatic potential in accordance with thisaspect of the present invention is illustrated by the followingdiscussion of uteroglobin protein, mRNA or DNA as an index of whetherlung tumors will metastasize.

[0085] METHODS

[0086] Uteroglobin as an Index of Metastasis

[0087] Lung cancer remains the major cause of cancer death among bothmales and females. Recognition of the expression of one or moreneoplastic antigens in advance of clinical cancer opens severalpotential therapeutic alternatives.

[0088] Four types of lung cancer are found in humans: squamous, adeno,small cell, and large cell. Each tumor expresses specificdifferentiation features or surface phenotype determinants, all of whichdistinguish these cells from normal cells. The development of monoclonalantibody diagnostic techniques has greatly enhanced the production ofreagents capable of differentiating normal cells from cancer cells anddifferentiating types of cancer cells from other cancer cells.

[0089] Unfortunately, it is difficult to distinguish benign fromcancerous tumors and, more importantly, slow growing localized tumorsfrom those formed by more aggressive, metastatic cancers. Thus, even thebest physician cannot accurately predict the course of progression of agiven lung cancer, and thus is unable to accurately prescribe the besttreatment regimen.

[0090] In one aspect the present invention overcomes this obstacle toeffective treatment of such tumors by providing a method of treatinglung tumors by administering an inhibitor of PLA₂ such as uteroglobin.

[0091] In another aspect, the present invention is also directed to amethod for inhibiting metastasis of lung epithelial tumor cells,comprising administering a therapeutically effective amount of aninhibitor of phospholipase A₂ to an animal.

[0092] In a further aspect, the present invention additionally providesfor a method for inhibiting invasion of lung epithelial tumor cells,comprising administering a therapeutically effective amount of aninhibitor of phospholipase A₂ to an animal.

[0093] An additional preferred aspect is a method for detecting oridentifying metastatic lung cancer, comprising: comparing an amount ofan inhibitor of phospholipase A₂ in a sample of lung epithelial tissueto at least one reference which correlates to the amount of inhibitor ofphospholipase A₂ in normal lung epithelial tissue, or to metastatic lungcancer, whereby differential amounts of the inhibitor of phospholipaseA₂ between the sample of lung epithelial tissue and the reference,detects or identifies metastatic lung cancer.

[0094] Another preferred aspect includes detecting or identifyingmetastatic lung cancer, comprising: assaying for inhibitor ofphospholipase A₂ in a sample of lung epithelial tissue; and comparingthe amount of the inhibitor in the sample to a reference whichcorrelates to the amount of the inhibitor in normal lung epithelialcells or metastatic lung epithelial cells, wherein differential amountsof the inhibitor of between the sample and the reference detects oridentifies metastatic lung cancer.

[0095] Another aspect detecting or identifying a pathological conditionof lung epithelial tissue, comprising: assaying for inhibitor ofphospholipase A₂ in a sample of lung epithelial tissue; and comparingthe amount of the inhibitor in the sample to a reference whichcorrelates to the amount of the inhibitor in normal lung epithelialtissue or metastatic lung epithelial cells, wherein differential amountsof the inhibitor between the sample and the reference detects oridentifies a pathological condition of lung tissue.

[0096] Yet another aspect includes a method for detecting or identifyingan inhibitor of phospholipase A₂ in a sample of lung epithelial tissue,comprising: assaying for an inhibitor of phospholipase A₂ in a sample ofsample of lung epithelial.

[0097] It is worth noting in this respect that previous studies did notidentify the relationship between metastatic potential of a tumor anddecreased expression of uteroglobin (or any other inhibitors ofarachidonic acid release). In previous studies, for instance,uteroglobin (called Clara cell 10 kDa protein, abbreviated CC10) wasused as a marker for certain types of cells, and cell-type specificityof its expression was studied. (As described in Linnoila et al.,A.J.C.P. 97(2): 235-243 (1992) and Peri et al., J. Clin. Invest. 92:2099-2109 (1993), which are incorporated by reference herein in theirentirety). In addition, CC10 expression was reported to vary betweenpatients and cell types. In particular, it was reported that CC10expression was lower in lung cancer patients and in smokers without lungcancer than it was in non-smokers, and decreased CC10 expression hasbeen loosely associated with neoplasm. (Broers et al., Lab. Invest. 66:337-346 (1992) and Jensen et al., Int. J. Cancer 58: 629-637 (1994),which are incorporated by reference herein in their entirety. However,no studies of CC10 expression have suggested that uteroglobin may beused to inhibit or reverse metastatic processes or that detecting thesudden lack uteroglobin expression in lung epithelial cells can be usedto gauge the metastatic stage or potential of those cells.

[0098] Specific Detection of Proteins

[0099] In preferred aspects of the invention, individual proteinsindicative of metastatic potential of tumors can be determined in cellsin biopsy material by conventional methods well known to those of skillin the art. In one aspect the inhibitor of phospholipase A₂ isuteroglobin, a mutein of uteroglobin, a peptide analog of uteroglobin ora mimetic of uteroglobin. Such methods are described in many standardtextbooks and laboratory manuals.

[0100] For instance, the techniques for making and using antibody andother immunological reagents and for detecting particular proteins insamples using such reagents are described in CURRENT PROTOCOLS INIMMUNOLOGY, Coligan et al., Eds., John Wiley & Sons, New York (1995). Asanother example, immunohistochemical methods for determining proteins incells in tissues are described in Volume 2, Chapter 14 of CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al., Eds., John Wiley & Sons,Inc. (1994). Finally, Linnoila et al., A.J.C.P. 97(2) : 235-243 (1992)and Peri et al., J. Clin. Invest. 92: 2099-2109 (1993).

[0101] For instance, the amount of uteroglobin in a sample can bedetermined in accordance with the invention by histochemical methods setout in Miyamoto et al., J. Urology 149: 1015-1019 (1993). As describedtherein, for instance, suitable biopsy material is obtained from apatient suspected of having benign lung hyperplasia or lung carcinomaand immediately placed into 0.01M phosphate buffered saline. Thereafter,the material is immediately processed. It is mounted on a brass plateusing rat liver homogenate as an adhesive. The material then is frozenin liquid nitrogen-cooled isopentane. Sections suitable for assay ofuteroglobin in cells of the material are sectioned in a cryostat.Sections are obtained across the biopsy material, avoiding parts of thebiopsy material that are damaged or deleteriously altered by the removalprocess.

[0102] Sections are dried at room temperature, fixed and then washed.Paraformaldehyde is a particularly useful fixative in this regard, butmany other fixatives also can be used. The sections may be pretreatedwith hydrogen peroxide and a non-ionic detergent, such as Triton X-100.Also, sections may be incubated with a blocking solution to reducenon-specific binding. For instance, the sections may be incubated withgoat blocking serum prior to incubation with a goat serum, goat antibodyor goat antibody-derived reagent.

[0103] Uteroglobin then is visualized for determination in the samplesusing a uteroglobin-specific binding reagent, such as a monoclonal or apolyclonal anti-uteroglobin antibody. Binding of theuteroglobin-specific reagent to cells in the sections may be determineddirectly, if the reagent has been conjugated to a detectable label, orusing a second or additional reagents, such as a secondaryantibody-enzyme conjugate.

[0104] In preferred embodiments of the invention, theuteroglobin-specific reagent is an antiserum, a polyclonal antibody, aderivative of a polyclonal antibody, a monoclonal antibody, a derivativeof a monoclonal antibody or an engineered antibody, such as a singlechain antibody. Derivatives of monoclonal and polyclonal antibodiesinclude conjugates and fragments. Antibodies conjugated to detectablelabels are preferred in this regard. Among detectable labels are enzymessuch as horseradish peroxidase. Among fragments preferred in this regardare Fab fragments, F(ab′)₂ fragments and F(ab′) fragments.

[0105] Sections are incubated with uteroglobin-specific reagent underconditions effective for the uteroglobin-specific reagent to bindefficiently to uteroglobin in said cells, while binding to othercellular components is inefficient; i.e., under conditions effective forthe ratio of specific to non-specific binding to provide accuratedetermination of uteroglobin content in cells of the biopsy material.

[0106] At the same time, control sections may be incubated under thesame conditions with a corresponding reagent that is not specific foruteroglobin, to estimate background binding. For polyclonal immuneserum, for instance, control sections can be incubated with preimmuneserum to monitor background, non-specific binding. After the incubationperiod, the specific reagent, and any reagent used in the controls, isremoved, as by washing.

[0107] If the primary, uteroglobin-specific reagent is detectablylabeled, then the label may be determined and, thereby the uteroglobincontent of cells in the sample. In this case, controls preferably wouldbe labeled and would be determined in like fashion. More often, andpreferably, a secondary reagent is used to visualize binding of reagentson the sections, as described below.

[0108] After removing unbound specific and non-specific reagents, testand control sections are incubated with a secondary reagent that bindsspecifically to the primary, uteroglobin specific reagent and itscounterpart in the controls. Preferably, the secondary reagent is abiotinylated anti-antibody.

[0109] The sections are incubated with the secondary reagent underconditions for the reagent to bind efficiently to the primary reagent(and its counterpart in the controls) in the cells, while binding toother cellular components is inefficient; i.e., under conditionseffective for the ratio of specific to non-specific binding to provideaccurate determination of uteroglobin content in cells of the biopsymaterial.

[0110] Thereafter, the unbound fraction of the secondary reagent isremoved from the sections. The secondary reagent, and its counterpart inthe controls, then is determined. If the secondary reagent comprises adetectable label, incubation with a tertiary reagent generally will notbe necessary. However, use of a tertiary reagent comprising a detectablelabel is more commonly employed for immunocytochemical analysis,generally. Therefore, for illustrative purposes, the three componentassay is described here.

[0111] In preferred aspects of the invention, the inhibitor is detectedusing ELISA immunoassay, radioimmunoassay, chemiluminescenceimmunoassay, fluorescence immunoassay, cell sorting assay, fluorescenceactivated cell sorting assay, Western blotting techniques,immunoprecipitation assay, colorimetric or densitometric assay,enzymatic assay, and immunostaining assay.

[0112] The sections then are incubated with a tertiary reagentcomprising a detectable label that binds specifically to the secondaryreagent. Incubation is carried out under conditions effective for thetertiary reagent to bind efficiently to the secondary reagent bound toprimary reagent in cells in the test sections or its counterpart incontrol sections, while binding to other cellular components isinefficient; i.e., under conditions effective for the ratio of specificto non-specific binding to provide accurate determination of uteroglobincontent in cells of the biopsy material. A preferred tertiary reagentcomprising a detectable label is an avidinated enzyme for binding tobiotinylated secondary reagent. A preferred enzyme in this regard ishorseradish peroxidase.

[0113] Unbound tertiary reagent is removed, by washing the sections withbuffer, for instance. The detectable label bound in cells in the biopsymaterial then may be determined. In preferred embodiments of theinvention, sections are incubated under conditions effective for anenzyme in the tertiary reagent to catalyze a chromogenic reaction.Binding of the uteroglobin-specific reagent is determined by the colorgenerated by the reaction.

[0114] Suitable reagents and conditions for carrying out thedetermination of uteroglobin in cells in biopsy samples are well knownand readily available. A multiplicity of procedures and reagents can beeffectively employed for this purpose. Such reagents and techniquesroutinely are employed by those of skill in the arts ofimmunocytochemistry, histopathology and cytology.

[0115] A particularly preferred embodiment of the invention relates tothe detection of aberrant uteroglobin protein. Surprisingly, an aberrantform of native uteroglobin is expressed and directly correlates to thedecrease in native or normal uteroglobin as metastasis progresses.Accordingly, one aspect of the present invention involves not only thedetection of the loss of normal uteroglobin as a gauge of metastatic orinvasive activity, but also the increase in the level of aberrantuteroglobin. This increase provides a positive detection whereas theloss of normal uteroglobin relies upon the absence. Furthermore, thisaberrant form of uteroglobin is secreted in sputum and bronchial fluid,as well as other related bodily fluids such as respiratory fluids andthe like where such proteins are found. Accordingly, a method and kitfor detection of an aberrant form of uteroglobin as a direct indicatorof metastasis and invasiveness is provided.

[0116] Kits for performing such assays, in whole and in part, are widelyavailable from numerous commercial suppliers. Incubation with secondaryantibodies, and subsequent visualization of uteroglobin, can carried outaccording the given procedures prescribed by commercial suppliers.

[0117] In preferred embodiments the sections are stained withhematoxylin and eosin to confirm pathology and to facilitate comparisonof uteroglobin in normal and diseased cells in the same section.

[0118] In particularly preferred embodiments, the relative staining ofdiseased and normal cells in a sections is compared with staining incontrol cells. The control cells are reference standards which typifyresults obtained by a given procedure in normal cells, cellscharacteristic of benign tumors, and cells characteristic of malignanttumors. Within any category, moreover, control cells may provide agraded or gradient series of characteristic standard or normal results.Uteroglobin in control cells may be determined at the same timeuteroglobin is determined in cells of the biopsy sample, or at anothertime. In a particularly preferred embodiment of the invention,uteroglobin is determined in control cells which serve as a standardreference series for subsequent clinical assays.

[0119] Normal tissue immunocytochemical techniques, such as thosedescribed above, reveal very heavy staining of uteroglobin in theluminal surface of lung epithelial cells. Biopsy samples that evidenceintermediary pathology thought to precede neoplasia, such asintraepithelial neoplasia, show a pattern of uteroglobin stainingsimilar but weaker than that of normal cells. Biopsy material frommalignant tumors shows significant decreases in staining of uteroglobinin cells. The decrease in staining of the luminal surface of epithelialcells in lung tumors is particularly dramatic. Whereas in normal tissuethe luminal surface of epithelial cells shows the highest staining foruteroglobin, uteroglobin staining either cannot be detected or is faintin the same cells in metastatic lung tissue.

[0120] It is this ability to differentiate between normal tissue,neoplastic conditions, and cancer which provides for the identificationof metastasis. Due to the association of UG with cancer, detectinghigh-grade cancer provides an early diagnosis, prognosis and treatmentof lung cancer.

[0121] Furthermore, it is further expected that a close relationshipbetween the age-related prevalence of neoplasia and the age-relatedprevalence of lung cancer with the occurrence of neoplasia mirroring theoccurrence of lung cancer but having a 20-30 year lag time. It is thisrelationship which provides a practitioner the diagnostic and prognosticability of early detection and treatment.

[0122] The risk of developing invasive cancer i.e. metastatic cancer isgauged by the decrease in uteroglobin in diseased cells in the biopsysample relative to cells in normal tissue of the same type, as describedabove. In biopsy material containing both normal and diseased tissue thestaining of cells in the normal and diseased tissue can be compared onthe same section. In general, the cells in low grade relatively confinedtumors express uteroglobin in amounts similar to normal cells. The cellsin aggressive, invasive tumors express little or no uteroglobin and arepoorly differentiated in their morphology.

[0123] Specific Detection of mRNA and DNA

[0124] mRNA also can be detected or assayed for in lung epithelial cellsof the biopsy samples to detect or identify metastatic processes ormetastatic potential. mRNA can be determined by a variety of methodswell known to those of skill in the art, which can be carried out usingwell known and readily available starting materials, including thosewidely available from commercial suppliers. Techniques useful in thisregard are described in the foregoing references. Techniques that may beparticularly pertinent in this regard relating to uteroglobin aredescribed in Broers et al., Lab. Invest. 66: 337-346 (1992) and Jensenet al., Int. J. Cancer 58: 629-637 (1994), incorporated herein asreferred to above.

[0125] A given mRNA may be assayed by in situ hybridization to aspecific probe. Such probes may be cloned DNAs or fragments thereof,RNA, typically made by in vitro transcription, or oligonucleotideprobes, usually made by solid phase synthesis. Methods for making andusing probe suitable for specific hybridization in situ are ubiquitouslyknown and used in the art.

[0126] By specific hybridization is meant that the probe forms a duplexwith the given, target mRNA that is stable to the conditions ofhybridization and subsequent incubations and that duplexes formedbetween the probe and other, non-target mRNAs are not stable andgenerally do not persist through subsequent incubations. Specifichybridization thus means that the ratio of hybridization to target andnon-target mRNAs provides an accurate determination of the target mRNAin cells in the biopsy sample.

[0127] In a particularly preferred embodiment of the present invention aprobe that hybridizes specifically to uteroglobin mRNA is used todetermine uteroglobin mRNA in cells of biopsy tissue, particularly lungepithelial cells.

[0128] Techniques suitable for in situ determination of target mRNAs,such as uteroglobin mRNA, are described in a variety of well known andreadily available laboratory manuals, as well as the primary literature.An illustrative procedure from the primary literature in this regard isdescribed in Broers et al. Laboratory Investigation 66 (3): 337-346(1992).

[0129] In general, biopsy material is obtained by suitable surgicalprocedure and snap frozen, as by freezing in methybutane/dry ice. Thesamples can be embedded and sectioned much as described above for thedetermination of protein is biopsy samples. Sections can be thawed ontoand affixed to glass slides previously cleaned with acid and ethanol andcoated with poly-L-lysine. The tissue sections thereafter can be exposedto buffered formaldehyde, acetylated, treated with buffered glycine andthen prehybridized in 50% formamide, 2X SSC (where 1X SSC is 0.15M NaCl,0.015M sodium citrate, pH 7.0). After prehybridization the sections canbe hybridized to the labeled probe in 50% formamide, 10% dextransulfate, 2X SSC.

[0130] The exact conditions of the steps in the procedure, especiallythe prehybridization, hybridization and criterion steps will be adjustedwith the T_(m) (or the T_(d)) of the probe and to provide the desireddegree of specificity of hybridization; i.e., the desired stringency.

[0131] Theoretical approximations and empirical methods for determiningproper conditions in this regard are well known and routinely practicedby those skilled in the pertinent arts. Approximation calculations andexperimental techniques in this regard are described, for instance, inSambrook et al. (1989) referred to herein above.

[0132] Those of skill will appreciate, for instance, that the formamidein the foregoing solutions serves to provide equivalent hybridizationconditions at lower temperature. For instance, hybridization in 50%formamide at about 50EC provides conditions similar to hybridization at65EC without formamide. The lower temperature of hybridization can helppreserve the biopsy sections during the hybridization procedure, aidingsubsequent identification and examination of cells and mRNA content.Other agents that preserve features of the tissue sections that aidanalysis likewise are preferred.

[0133] Dextran sulfate generally is used to accelerate the hybridizationreaction and to drive it to completion in a shorter period of time, asis well known. Similar agents that increase the rate of hybridization,consistent with accurate determination of specific mRNA content, alsoare useful in the present invention.

[0134] Following hybridization, the probe-containing solution andunbound probe are removed. Typically, the sections are washed severaltimes with prehybridization buffer, such as 50% formamide, 2X SSC, at orslightly above the hybridization temperature.

[0135] If an RNA probe is used for detection of the target mRNA, thesections then are treated with RNAseA, typically in the same solution,and then washed to remove RNAseA and byproducts with 50% formamide, 2XSSC under the same conditions as the previous washings.

[0136] Finally, the sections typically are washed several additionaltimes in 2X SSC at room temperature and then air dried.

[0137] Radioactive probes generally are visualized by autoradiography.For this purpose slides can be dipped in a photographic emulsion, driedand allowed to expose the emulsion at 4EC for an appropriate period oftime. Using a preferred emulsion, NTB-2 nuclear track emulsion, exposuretimes of 3 to 7 days are appropriate. The exposure time can be alteredby a variety of factors including the use of more highly labeled probes.

[0138] The emulsions are developed at the end of the exposure period andthen, typically, counterstained with hematoxylin and eosin.Subsequently, labeling of target mRNA in cells can be assessed bymicroscopy using brightfield and darkfield illumination.

[0139] As discussed above regarding protein, the abundance anddistribution of the target mRNA in cells in the biopsy section indicateswhether tumor cells are metastatic and invasive or have the potential tometastasize. Particularly, the relative abundance of mRNA in diseasedand normal cells indicates metastatic potential.

[0140] A variety of controls may be usefully employed to improveaccuracy in assays of this type. For instance, sections may behybridized to an irrelevant probe and sections may be treated withRNAseA prior to hybridization, to assess spurious hybridization.

[0141] Thus, for instance, as discussed for uteroglobin protein, normaltissue exhibits high concentrations of uteroglobin mRNA in the lungepithelial cells. Intermediary pathology shows a lower concentration ofuteroglobin mRNA similar to but less than the hybridization to auteroglobin mRNA-specific probe exhibited by normal cells. Biopsymaterial from malignant tumors shows significant decreases in theconcentration of mRNA uteroglobin in cells by exhibiting little or nohybridization to a uteroglobin mRNA-specific hybridization probe.

[0142] Again, the differentiation between normal tissue, neoplasia, andcancer provides for the identification of and early diagnosis, prognosisand treatment of lung cancer.

[0143] Aberrant mRNA or DNA

[0144] Some metastatic tissues of lung origin exhibit seemingly normalhybridization to a uteroglobin-specific probe, even though cells in thesame tissue do not synthesize much, if any, uteroglobin protein. Thesecells typically exhibit aberrant uteroglobin mRNA, rather decreaseduteroglobin mRNA.

[0145] Splicing and other aberrations in mRNAs of cells of metastatictissue can be determined by northern and southern blotting techniquesand by PCR techniques. These techniques also are well known to those ofskill in the art and can be applied readily to the determination of mRNAin cells of biopsy material in accordance with the present invention.

[0146] Techniques employed to assess restriction fragment lengthpolymorphisms (“RFLP”) can be applied to detect some mutationsassociated with aberrant splicing patterns. The assessment always can bemade on the mRNA, but in some dysfunctions it can be made on the genomicDNA as well. The mRNA or DNA can be amplified prior to RFLP analysis, aswell, using PCR or other suitable technique.

[0147] In addition to RFLP techniques, SSCP can be used to detectaberrant splicing of messages, such as uteroglobin-specific mRNA. Forthis purpose, a target mRNA, such as uteroglobin mRNA, is amplified byreverse transcriptase-mediated PCR. The double-stranded amplified DNA isdenatured and run on gels in which mobility is quite sensitive to smallchanges in secondary structure.

[0148] Yet another technique that can be employed to determine aberrantsplicing, among other things, is ligase mediated PCR. This techniquealso is well known to those of skill in the art, and techniques suitableto the analysis and determination of mRNAs and genomic aberrations thathave been described in the literature readily can be applied to thedetermination of aberrant mRNAs in cells of tumor.

[0149] Biopsy material is prepared for the detection of mRNA and genomicDNA in epithelial cells. Particularly preferred in this regard is lungbiopsy material. Other preferred aspects of the invention includesamples of lung epithelial tissue is selected from the group consistingof lung biopsy, sputum, bronchial fluid, bronchial aspirate, and pleuralfluid.

[0150] Accordingly, probes specific for uteroglobin mRNA or foraberrations of uteroglobin mRNA or uteroglobin-encoding DNA indicativeof altered expression of uteroglobin may be used as hybridizationtargets for detecting the potential of lung epithelial cell tumors tometastasize.

[0151] CONTROL

[0152] Control cells may be developed to guide interpretation ofresults. In this regard, a protein or mRNA in accordance with theforegoing may be determined in biopsy material from representativetumors of a specific type, characteristic of a specific degree ofmetastatic potential.

[0153] Another preferred aspect includes a series of standards whichindicate an amount of the inhibitor of phospholipase A₂ and provides agradient from lower amounts to higher amounts of the inhibitor, whereinhigher amounts of inhibitor indicates normal lung epithelial cells andlower amounts of inhibitor indicates metastatic lung cancer.

[0154] Characterization in this regard may benefit from hindsight,following the actual course of tumor progression in patients as theyundergo treatment and thereafter. Control results characterizing agraded series of metastatic potential also may obtained from cellculture studies, as described elsewhere herein, illuminated in theexamples below.

[0155] The determination of a protein or mRNA, or other agent, as setout above, in a variety of tumors of known metastatic characteristic,and the correlation of the determinations with metastatic potential isanother preferred embodiment of the present invention.

[0156] KITS

[0157] Reagents for carrying out the methods described above may beincorporated into kits for use in treating metastatic lung cancer, anddetecting or identifying the metastatic potential of lung epithelialcells. All of the techniques and reagents discussed below may beincluded in these kits including reagents and methods set out in theexamples.

[0158] The kits also may include one or more control results, such asreference slides of immunocytochemical results characteristic of atumors with high and low metastatic potential, or reference slides of insitu hybridization results characteristic in the same regard. Preferablein this regard are kits that include a control series for interpretingresults. This control or standard may be in the form of written,graphic, or audiovisual presentation. For instance, one or morephotographs may be depict results from normal and/or metastatic tissue.In other ways, written descriptions, color charts, computerized imagesand sampling may be used. In addition, the control may be highlytumor-type-specific or it may be applicable to related types of tumors.

[0159] ANTI-METASTATIC AGENTS AND METHODS

[0160] The invention disclosed herein provides agents and methods forinhibiting or preventing metastasis. The following discussionillustrates the invention in this respect.

Anti-metastatic agents

[0161] In accordance with this aspect of the invention, proteins thatinhibit arachidonic acid release by cells of a tumor of epithelial cellorigin in an organism are administered by a route and in an amounteffective to prevent or inhibit metastasis of the tumor.

Inhibitors of arachidonic acid release

[0162] Without being limited to any theory of the invention, applicantsnote that arachidonic acid is a substrate in the synthetic pathway ofeicosanoids in cells. Various eicosanoids play a role in stimulating orinhibiting shape, attachment, motility and proliferation of cells. Insome aspects of the invention, inhibiting arachidonic acid release incells of tumors of epithelial cell origin inhibits or extinguishesmetastatic potential.

Inhibitors of phospholipase A₂

[0163] Compounds that inhibit phospholipase A₂ (PLA₂) are preferredcompounds of the present invention. PLA₂ is a membrane signaling enzymeof the arachidonic acid cascade, the series of enzymes, substrates,products and co-factors involved in the production and secretion ofarachidonic acid, and it generally will be the case that inhibitors ofPLA₂ activity generally will inhibit release of arachidonic acid.Notably, PLA₂ has been associated with processes of inflammation, ratherthan tumorigenesis or metastasis, and it has been suggested as a targetfor the control of chronic inflammation, but not as a target fordeveloping an anti-metastatic agent. Nevertheless, the present inventionprovides compositions and methods of PLA₂ inhibitors for inhibitingmetastasis of tumors of epithelial cell origin. The formulation and useof these compounds in the invention is illustrated by reference to thepreferred embodiments discussed below.

[0164] Especially preferred among PLA₂ inhibitors are uteroglobins,muteins of uteroglobins and peptide analogs of uteroglobin.Uteroglobins, muteins of uteroglobins and peptide analogs of uteroglobinare particularly preferred. Most particularly preferred is humanuteroglobin. The discussion below, directed to uteroglobin, particularlyhuman uteroglobin, illustrates the invention is this regard.

Uteroglobins

[0165] Uteroglobin, also called blastokinin, was first discovered as amajor protein component of the rabbit uterine fluid during earlypregnancy. The human counterpart to rabbit uteroglobin was first foundin nonciliated Clara cells in the distal bronchiole airway and wasoriginally designated Clara cell 10 kD protein, abbreviated as “CC10.”Uteroglobin also has been detected in humans in the uterus, respiratorytract, and lung gland, by immunohistochemical methods.

[0166] The complementary DNA for human uteroglobin (CC10) has beencloned and its sequence has been determined, as reported in Singh etal., BBA 950: 329-337 (1988), incorporated by reference herein in itsentirety.

[0167] Uteroglobin has been purified to homogeneity by at least twogroups and it has been structurally and functionally characterized inconsiderable detail. In brief, uteroglobin occurs in rabbits as a dinerof two identical chains. The monomers are 70 amino acids long. They arearranged antiparallel to one another in the dimer. Also, in the dimerthey are covalently linked by two symmetrical disulfide bonds, formedbetween ‘Cys-3’ and “Cys-69” and, reciprocally, between ‘Cys-69’ and“Cys-3” (where ‘ designates one chain in the dimer and “ designates theother chain). Each monomer chain contains four α-helical segments and aβ-turn, the later at Lys-26 to Gln-29. The structure, function andactivities of uteroglobin has been reviewed, for instance, by Miele etal., Endocrine Reviews 8: 474-490 (1987), which is incorporated byreference herein in its entirety.

[0168] Uteroglobin inhibits the activity of PLA₂, as shown by in vitroassays. Generally, it has been thought to have immunomodulatory oranti-inflammatory activities, or both, that act to protect the wetepithelia of organs that communicate with the external environment.Uteroglobin expression is steroid-sensitive and its secretion in theendometrium has been shown to be stimulated by progesterone. Uteroglobinalso has been reported to have an anti-chemotactic effect on neutrophilsand monocytes. Uteroglobin has not been seen as playing a role in canceror metastasis. Thus, it was surprising to find that uteroglobin, inaccordance with the present invention, can be used to inhibit or preventmetastasis of a tumor of epithelial cell origin in an organism.

[0169] Without being bound to any theory of the mechanism by whichuteroglobin inhibits metastasis, it appears, as the Examples show, thatthe inhibitory action of uteroglobin on metastasis results frominhibition of PLA₂ activity and inhibition of arachidonic acid releaseby the tumor cells.

[0170] Any uteroglobin that inhibits arachidonic acid release or thatinhibits or prevents metastasis of a tumor of epithelial cell origin maybe useful. Uteroglobins for use in the invention may be recovered fromnatural sources, may be made by recombinant means, may be produced bychemical techniques, may be made by semi-synthetic methods or may beobtained by a combination of techniques.

[0171] Methods for purifying uteroglobin to homogeneity from a naturalsource have been described in Nieto et al., Arch. Biochem. Biophys.180:80-92 (1977), which is herein incorporated by reference in itsentirety. Other methods for this purpose can be equally useful in thisregard.

[0172] The most highly preferred uteroglobin for use in the invention atthe present time is human uteroglobin. Preferably, human uteroglobin foruse in the invention is made by expression of a cloned gene in a hostcell in culture or in an animal. Techniques for expressing uteroglobinin this way are well known to those of skill in the art.

[0173] A cDNA encoding human uteroglobin, useful toward this end, hasbeen isolated, sequenced and expressed in cells in culture. Methods forexpressing cloned DNAs that encode uteroglobin have been describedspecifically with regard to human uteroglobin in Mantile et al., J. BiolChem. 268: 20343-20351 (1993) and Miele et al., J. Biol. Chem. 265:6427-6435 (1990), which, as noted below, are incorporated by referenceherein in their entirety.

[0174] Techniques for obtaining, manipulating and expressing clonedgenes to obtain uteroglobin for use in the present invention are wellknown to those of skill in the art and are described in protocol-likedetail in a variety of laboratory manuals. For instance, such methodsare set forth in Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2ND Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989), the entirety of which is herein incorporated byreference.

Muteins and peptide analogs

[0175] Techniques such as those described in the foregoing manual can beused to make variants and analogs of uteroglobin and other proteinsuseful in the invention. Recombinant DNA methods, chemical syntheticmethods, enzymatic methods and mixed methods for making, altering andutilizing muteins and peptide analogs are well known and are describedhere only briefly to illustrate their applicability to the presentinvention.

[0176] muteins

[0177] It will be appreciated by those of skill that cloned genesreadily can be manipulated to alter the amino acid sequence of aprotein. The cloned gene for human uteroglobin can be manipulated by avariety of well known techniques for in vitro mutagenesis, among others,to produce variants of the naturally occurring human protein, hereinreferred to as muteins, that may be used in accordance with theinvention.

[0178] The variation in primary structure of muteins of uteroglobinsuseful in the invention, for instance, may include deletions, additionsand substitutions. The substitutions may be conservative ornon-conservative. The differences between the natural protein and themutein generally conserve desired properties, mitigate or eliminateundesired properties and add desired or new properties. In the presentinvention the muteins generally are those that maintain or increaseanti-metastatic activity. Particularly, uteroglobin muteins are aminoacid sequence variants of uteroglobin that maintain or increase theanti-metastatic activity of uteroglobin.

[0179] peptide analogs

[0180] Similarly, techniques for making small oligopeptides andpolypeptides that exhibit activity of larger proteins from which theyare derived (in primary sequence) are well known and have become routinein the art. Thus, peptide analogs of proteins of the invention, such aspeptide analogs of uteroglobin that exhibit anti-metastatic activityalso are useful in the invention.

Mimetics

[0181] Mimetics also can be used in accordance with the presentinvention to prevent or inhibit metastasis of tumors. The design ofmimetics is known to those skilled in the art, and is generallyunderstood to be peptides or other relatively small molecules that havean activity the same or similar to that of a larger molecule, often aprotein, on which they are modeled. Thus, by way of illustration,uteroglobin mimetics, for instance, can be used in accordance with thepresent invention in the same manner as uteroglobin itself, to preventor inhibit metastasis of a tumor.

[0182] The design of such mimetics can be based on thestructure-function relationship of uteroglobin. By studying the effectof mutations on anti-metastatic activity of uteroglobin the sites in theprotein responsible for anti-metastatic activity can be identified. Invitro mutagenesis procedures that can be used to systematically altercloned genes, such as cDNAs encoding uteroglobin and other proteins withanti-metastatic activity of the present invention, are described inSambrook et al. (1988). Systematically mutagenized proteins, alsoreferred to as muteins as noted elsewhere herein, can be produced usingsuch altered DNA by standard methods for expression cloned genes inorganisms to produce heterologous proteins. Such methods are well knownto those of skill and are described in, for instance, Sambrook et al.(1988) referred to herein above. The muteins so produced then can beassayed for anti-metastatic activity, using in vitro or in vivo assaysthat model or measure metastatic activity. Suitable methods aredescribed herein and illustrated in the examples below.

[0183] Methods for determining aspects of protein structure also arewell known to those of skill in the art. To some extent, the structureof a given protein can be approximated by analogy to structures ofrelated proteins. Physical and chemical information about a proteinstructure can be obtained by a wide variety of well known techniques,including active site modification techniques, NMR, and X-raycrystallography.

[0184] This information can be combined with information from studiesthat correlate structural alterations with changes in activity, such asthe mutagenesis studies described above, to generate a map of the shapeand chemical functions important to a given activity of a protein.

[0185] Molecules that mimic the shape and chemical functionality thatprovide the desired activity, then can be designed and synthesized.Computer modeling methods that can be employed toward this end, as wellas methods of organic synthesis, peptide synthesis and for the synthesisof other classes of compounds that can be used to produce mimetics inaccordance with this aspect of the invention are well known to those ofskill in the art.

[0186] Once a mimetic has been designed and synthesized, it can beassayed for anti-metastatic activity using techniques for this purpose,such as those described elsewhere herein.

[0187] Results of activity studies and of structural studies of themimetics themselves can be used to design further mimetics that are moreeffective, have fewer undesirable side effects, or have additionalactivities, such as by combining two mimetics in a single molecule.

[0188] In the same manner as for uteroglobin, mimetics can be designedfor other compounds that have anti-metastatic activity. Preferred inthis regard, as described above, as anti-metastatic mimetic compoundsthat inhibit arachidonic acid release by cells of a cancer of epithelialcell origin. Particularly preferred are mimetics that inhibitphospholipase A₂ in such cells. In this regard, mimetics of uteroglobinare especially preferred. Among the most highly preferred mimetics inthis regard are mimetics of human uteroglobin.

Small molecule drugs

[0189] Compounds other than the proteins, muteins, protein-derivedpeptides, mimetics and the like discussed above that inhibit arachidonicacid release by cells of cancers of epithelial cell origin also may beuseful in the present invention. Among such compounds are certain smallorganic molecules, which may be mimetics, that inhibit arachidonic acidrelease. Inhibition may be mediated by inhibition of PLA₂ activity, orby inhibition of other enzymes or intermediates involved in metabolicinteractions that result in arachidonic acid release.

[0190] Among such compounds is the anti-inflammatory agent mepacrine,which has been shown to inhibit PLA₂, and an experimental drug,indomethacin, which has been shown to inhibit cyclooxygenase. Bothcompounds exhibit anti-metastatic activity in in vitro assays, at dosesthat have been shown to be non-toxic in patients. Both compounds, thus,can be utilized in accordance with the present invention.

[0191] PLA₂, as noted herein above, is a key enzyme in the arachidonicacid cascade. As noted above, inhibitors of PLA₂ are most preferred inthe invention, in this regard. Among small molecule drugs, mepacrine ispreferred among inhibitors of PLA₂. Other relatively small moleculedrugs (small, in this case, meaning small relative to proteins ofaverage size) that, like mepacrine, inhibit PLA₂ also will be useful inthe invention, in the same fashion as mepacrine and the other PLA₂inhibitors discussed herein above. Among small molecule drugs, such PLA₂inhibitors are particularly preferred. In this regard, mepacrine ishighly preferred and other compounds that are similar to mepacrine inchemical structure are particularly preferred.

[0192] Cyclooxygenase is the key enzyme in the cyclooxygenase-dependentpathway of arachidonic acid metabolism, wherein arachidonic acid is aprecursor in the synthesis of prostaglandins, prostacyclins andthromboxanes. Among small molecule drugs, inhibitors of cyclooxygenasealso are preferred for use in the invention disclosed herein.Nonsteroidal anti-inflammatory agents (“NSAIDs”) are among the smallmolecule drugs, as the term is used herein, that inhibit cyclooxygenaseand are preferred in the invention in this regard. NSAIDs are described,for instance, in PRINCIPLES OF PHARMACOLOGY, Munson et al., EDs.,Chapman & Hall, New York (1995), which is incorporated herein byreference in part pertinent thereto, including, particularly, Chapter74.

[0193] Among NSAIDs in accordance with this aspect of the invention areaspirin, phenylbutazone, ibuprofen, sulfinpyrazone (Anturane) andindomethacin. In this regard, indomethacin is particularly preferred andcompounds that are similar to indomethacin in chemical structure alsoare preferred.

[0194] Lipoxygenase is the key enzyme in the lipoxygenase-dependentpathway of arachidonic acid metabolism, wherein arachidonic acid is aprecursor in the synthesis of thromboxanes. Inhibitors of lipoxygenase,and other downstream enzymes of the lipoxygenase-dependent pathway alsomay be of use in the present invention.

[0195] Cancers

[0196] The methods and kit of the present invention may be applied tothe treatment of a variety of cancers of epithelial cell origin. Amongthese are metastatic cancers of breast, lung, colon, bladder, lung,gastrointestinal track, endometrium, tracheal-bronchial tract, pancreas,liver, uterus, nasopharynges and the skin. An especially preferredtarget is lung cancer, particularly lung cancer of epithelial cellorigin.

[0197] The following detailed discussion of lung cancers is provided inillustration of the compositions and methods of the invention not onlyas to lung cancers, but also other cancers that may be treated inanalogous or identical fashion, in accordance with the presentinvention.

Lung adenocarcinoma

[0198] Lung cancer remains the major cause of cancer death among bothmales and females. Recognition of the expression of one or moreneoplastic antigens in advance of clinical cancer opens severalpotential therapeutic alternatives.

[0199] With lung cancer, as with all solid tumors, it is the metastaticencroachment of the tumor on other vital function that causes the demiseof the patient. Approximately 10% of patients are diagnosed initiallywith metastatic disease. Ultimately, 30-40% of patients with this cancerwill develop metastatic disease. Once metastasis occurs there is a thecancer follows a relentless progression.

[0200] Invasion is a prerequisite for migration of tumor cells. Inconnective tissue, stroma and basement membranes form the major physicalbarriers to the migration process. Invasion of the local extracellularmatrix (ECM) by tumor cells thus can be marked as the first step inmetastasis. The sequential biochemical mechanism of ECM invasion firstinvolves cell attachment to specific components of ECM followed by aprogressive cascade of proteolytic dissolution. Lung cancers which growto a critical size exhibit extracapsular invasion and metastasize tospecific anatomical sites apparently in response to stromal cellsecretory proteins which are necessary for their growth andproliferation. Invasive migration of tumor cells within the lung mayoccur as a function of chemokinesis along anatomical paths of leastresistance, such as the perineural duct. Further establishment ofmetastasis relies upon successful penetration of the circulatory orlymphatic system, followed by vessel extravasation at the secondaryorgan, which frequently is bone tissue for cancers originating from thelung. Nearly all of these steps, including attachment, matrixdegradation and migration, can be modeled experimentally in vitro bymeasuring invasion of a reconstituted basement membrane (RBM) barrier inresponse to fibroblast-conditioned medium (FCM) which serves as achemo-attractant.

[0201] In vivo growth and proliferation of lung tumor cells primarily isresponsive to stromal cell (fibroblast) secretory proteins.Extracapsular invasiveness of lung tumor cells can be modeled bymigration of tumor cells in vitro into reconstituted basement membrane(RBM) in the presence and absence of a chemo-attractant, such asfibroblast conditioned medium (FCM). The assay determines cells thathave attached to the RBM, degraded the RBM enzymatically and, finally,cells that have towards the FCM side of the membrane. The events in thein vitro invasion assay comport with the important steps observed in themetastatic spread of tumor cells through the basement membrane in vivo.

[0202] Lung tumors frequently initially metastasize to regional lymphnodes, having disseminated through the lymphatic circulation. They alsospread to other sites through the vascular system, which is extensivelyinterconnected to the lymphatics. The final site of formation ofmetastasis is a function of a number of parameters, including: (i) thefirst capillary bed encountered by blood vessels draining the tumor, and(ii) organ preference of the tumor cells with respect to characteristicsof specific tissues that nurture attachment and growth of tumor cellswith metastatic potential.

[0203] Metastatic potential of lung cancers of epithelial cells origincan be inhibited by methods of the invention. In particular, metastasisof these cancers can be inhibited by human uteroglobin, as shown by theexamples set out herein below.

[0204] Route of Administration

[0205] Therapeutic treatment with uteroglobin can utilize any type ofadministration including topical, other non-invasive and invasive means.

[0206] Administration by non-invasive means may be by oral, intranasalor transdermal routes, among others.

[0207] Generally, at the present time, invasive techniques arepreferred. Administration by invasive techniques may be intravenous,intraperitoneal, intramuscular or directly in tumors, among others.

[0208] Administration may be by a single dose, it may be repeated atintervals or it may be continuous. Since uteroglobin is small, easilydiffusible, and relatively stable it is well suited to long-termcontinuous administration, such as by perfusion pump. Where continuousadministration is applied, infusion is preferred. In this situation,pump means often will be particularly preferred for administration.Especially, subcutaneous pump means often will be preferred in thisregard.

[0209] In other situations it will be desirable to administereduteroglobin and other agents of the present invention by intramuscularself-injection on a regular basis.

[0210] Compositions and methods of the invention also may utilizecontrolled release technology. Thus, for example, uteroglobin may beincorporated into a hydrophobic polymer matrix for controlled releaseover a period of days. Such controlled release films are well known tothe art. Examples of polymers commonly employed for this purpose thatmay be used in the present invention include nondegradableethylene-vinyl acetate copolymer and degradable lactic acid-glycolicacid copolymers. Certain hydrogels such aspoly(hydroxyethylmethacrylate) or poly (vinylalcohol) also may beuseful, but for shorter release cycles then the other polymer releasessystems, such as those mentioned above.

[0211] Dose

[0212] The quantity of the active agent for effective therapy willdepend upon a variety of factors, including the type of cancer, means ofadministration, physiological state of the patient, other mendicantsadministered, and other factors.

[0213] Treatment dosages generally may be titrated to optimize safetyand efficacy. Typically, dosage-effect relationships from in vitrostudies initially will provide useful guidance on the proper doses forpatient administration. Studies in animal models also generally may beused for guidance regarding effective dosages for treatment ofmetastatic cancers in accordance with the present invention.

[0214] These considerations, as well as effective formulations andadministration procedures are well known in the art and are described instandard textbooks, such as GOODMAN AND GILMAN'S: THE PHARMACOLOGICALBASES OF THERAPEUTICS, 8th Ed., Gilman et al. Eds. Pergamon Press (1990)and REMINGTON'S PHARMACEUTICAL SCIENCES, 17th Ed., Mack Publishing Co.,Easton, Pa. (1990), both of which are incorporated by reference hereinin their entirety.

[0215] Typical therapeutic doses will be about 0.1 to 1.0 mg/kg of bodyweight of pure uteroglobin. The does may be adjusted to attain,initially, a blood level of about 0.1 μM.

[0216] A particular formulation of the invention uses a lyophilized formof uteroglobin, in accordance with well known techniques. For instance,1 to 100 mg of uteroglobin may be lyophilized in individual vials,together with carrier and buffer compound, for instance, such mannitoland sodium phosphate. The uteroglobin may be reconstituted in the vialswith bacteriostatic water and then administered, as described elsewhereherein.

[0217] Administration Regimen

[0218] Any effective treatment regimen can be utilized and repeated asnecessary to affect treatment.

[0219] In clinical practice, the compositions containing uteroglobin orrecombinant uteroglobin, alone or in combination with other therapeuticagents are administered in specific cycles until a response is obtained.

[0220] For patients who initially are not suffering from metastaticdisease, uteroglobin-based drugs can be used as an immediate initialtherapy prior to surgery and radiation therapy, and as a continuouspost-treatment therapy in patients at risk for recurrence or metastasis(based upon high markers, locally extensive disease, and/or pathologicalevidence of tumor invasion in the surgical specimen). Therapy for thesepatients aims, for instance, to decrease the escape of potentiallymetastatic cells from the primary tumor during surgery or radiotherapy,decrease the escape of tumor cells from undetectable residual primarytumor, decrease tumor cell attachment to the interior vessel wall,decrease the migration of tumor cells out of the vessel, and therebydecrease invasion into the interstitial spaces of the distal organ.

[0221] For patients who are afflicted with metastatic disease,uteroglobin-based drugs can be used as a continuous supplement to, orpossible as a replacement for hormonal ablation. A goal of therapy forthese patients is to slow tumor cell escape from both the untreatedprimary tumor and from the existing metastatic lesions in order to slowthe progressive encroachment of further metastases.

[0222] In addition, the invention may be particularly efficacious duringpost-surgical recovery, where the present compositions and methods maybe particularly effective in lessening the chances of recurrence of atumor engendered by shed cells that cannot be removed by surgicalintervention.

[0223] Gene Therapy

[0224] Certain embodiments of the present invention relate toanti-metastatic gene therapy. Gene therapy is a new approach totreatment of diseases. Currently, gene therapy protocols relate totherapy of certain carefully chosen disorders, including certaininherited disorders, a number of aggressively fatal cancers and AIDS.The restricted application of gene therapy to a few disorders reflectsconcerns about the efficacy, safety and ethical implications of theapproach in general, and current techniques in particular. Despite thecautious approach mandated by these concerns, and despite the fact thattechniques for carrying out gene therapy are still in an early stage ofdevelopment, results from the first few trials have been veryencouraging, some spectacularly so. It seems certain that gene therapytechniques will improve rapidly and that gene therapies soon willdevelop into an increasingly important and ubiquitous modality fortreating disease. (Reviewed, for instance, in Tolstoshev, Ann. Rev.Pharm. Toxicol. 32: 573-596 (1993) and Morgan et al., Ann. Rev. Biochem.62: 191-217 (1993), which are incorporated by reference herein in theirentirety).

[0225] The delivery of a variety of therapeutic agents clearly will beaccomplished by gene therapy techniques. Many of the procedures now inuse or under current development for gene therapy may be used inaccordance with the present invention to prevent or inhibit metastasis.Additional techniques that will be developed in the future similarlywill be found useful in the present invention. The following discussionis illustrative of the use of gene therapy techniques to prevent orinhibit metastasis in accordance with the present invention.

[0226] By gene therapy, in the following discussion, generally is meantthe use of a polynucleotide, in a cell, to achieve the production of anagent and the delivery of the agent to a tumor in situ, i.e., in apatient, to engender an anti-metastatic effect. The agent may itself bea anti-metastatic agent or it may engender the production of ananti-metastatic agent upon introduction into the patient.

[0227] Approaches to genetic therapy currently being developed, whichcan be used in accordance with this aspect of the invention disclosedherein, often are grouped into two major categories: ex vivo and in vivotechniques.

[0228] Ex vivo techniques generally proceed by removing cells from apatient or from a donor, introducing a polynucleotide into the cells,usually selecting and growing out, to the extent possible, cells thathave incorporated, and, most often, can express the polynucleotide, andthen introducing the selected cells into the patient. Cells that targettumor cells in vivo, including tumor cells that have migrated fromprimary or secondary tumor sites, generally are preferred in this typeof gene therapy.

[0229] In addition, as described further below, the polynucleotide maybe introduced directly into the patient. The polynucleotide in this casemay be introduced systemically or by injection into a tumor site. Thepolynucleotide may be in the form of DNA or RNA, alone or in a complex,or in a vector, as discussed further below.

[0230] The polynucleotide may be in any of a variety of well-knownforms, for instance, a DNA, a DNA fragment cloned in a DNA vector, a DNAfragment cloned in DNA vector and encapsidated in a viral capsid.

[0231] The polynucleotide may be an RNA or a DNA. More typically it is aDNA. It may include a promoter, enhancer and other cis-acting controlregions that provide a desired level and specificity of expression inthe cells of a region operably linked thereto that encodes an RNA, suchas an anti-sense RNA, or a protein. The polynucleotides may containseveral such operably linked control and encoding regions for expressionof one or more mRNAs or proteins, or a mixture of the two.

[0232] Preferred in this regard are polynucleotides that encode theanti-metastatic agents described herein above. As noted in the foregoingdiscussion, inhibitors of arachidonic acid release are preferred.Inhibitors of PLA₂ activity are particularly preferred. Among PLA₂inhibitors, uteroglobins are particularly preferred, human uteroglobinparticularly among uteroglobins.

[0233] Muteins and polypeptide analogs of protein inhibitors also areuseful in the invention and may be encoded by polynucleotides for genetherapy to inhibit metastasis. In this regard, muteins and polypeptideanalogs of the foregoing preferred embodiments also are preferred inthis aspect of the invention.

[0234] In addition, peptide mimetics that can be encoded by apolypeptide for synthesis in cells can be used in accordance with thisaspect of the invention. Preferred embodiments in this regard those setout above.

[0235] The polynucleotide may be introduced into cells either ex vivo orin vivo, including into the tumor in situ. A variety of techniques havebeen designed to deliver polynucleotides into cells for constitutive orinducible expression, and these routine techniques can be used in genetherapy of the present invention as well. Polynucleotides will bedelivered into cells ex vivo using cationic lipids, liposomes or viralvectors. Polynucleotides will be introduced into cells in vivo,including into cells of tumors in situ, using direct or systemicinjection. Methods for introducing polynucleotides in this manner caninvolve direct injection of a polynucleotide, which then generally willbe in a composition with a cationic lipid or other compound or compoundsthat facilitate direct uptake of DNA by cells in vivo. Such compositionsmay also comprise ingredients that modulate physiological persistence.In addition, polynucleotides can be introduced into cells in vivo inviral vectors.

[0236] Genetic therapies in accordance with the present invention mayinvolve a transient (temporary) presence of the gene therapypolynucleotide in the patient or the permanent introduction of apolynucleotide into the patient. In the latter regard, gene therapy maybe used to repair a dysfunctional gene to prevent or inhibit metastasis.

[0237] Genetic therapies, like the direct administration of agentsdiscussed above, in accordance with the present invention may be usedalone or in conjunction with other therapeutic modalities.

[0238] Combined with Other Treatments

[0239] Uteroglobin may be used in conjunction with other treatmentmodalities. Other common treatment modalities are discussed belowspecifically by reference to lung cancer. It will be appreciated thatsimilar consideration will apply to treatment of other metastaticcancers. The present invention may be used in conjunction with anycurrent or future therapy.

[0240] Surgery and Radiation

[0241] In general, surgery and radiation therapy are employed aspotentially curative therapies for patients under 70 years of age whoare afflicted with clinically localized disease and are expected to liveat least 10 years. Neither treatment modality has a significant role inthe management of metastatic diseases, and neither treatment isgenerally performed if metastasis is present at initial diagnosis.

[0242] Histopathological examination of surgical specimens reveals thatapproximately 63% of patients undergoing surgery (40% of total patients)have locally extensive tumors or regional (lymph node) metastasis thatwas undetected at initial diagnosis. These patients are at asignificantly greater risk of recurrence or metastasis. Approximately40% of these patients will actually develop recurrence or metastasiswithin 5 years after surgery. Results after treatment with radiation areeven less encouraging. Approximately 80% of patients who have undergoneradiation as their primary therapy have disease persistence or developrecurrence or metastasis within 5 years after treatment.

[0243] Currently, surgical and radiotherapy patients generally do notreceive any immediate follow-up therapy. Rather, they typically aremonitored for elevated Prostate Specific Antigen (“PSA”), which is theprimary indicator of recurrence or metastasis.

[0244] Thus, there is considerable opportunity to use the presentinvention in conjunction with surgical intervention.

[0245] Hormonal Therapy

[0246] Hormonal ablation is the most effective palliative treatment forthe 10% of patients having metastatic disease at initial diagnosis.Hormonal ablation by medication and/or orchiectomy is used to blockhormones that support the further growth and metastasis of lung cancer.With time, both the primary and metastatic tumors of virtually all ofthese patients become hormone-independent and resistant to therapy.Approximately 50% of patients with metastatic diseases die within 3years after initial diagnosis, and 75% of such patients die within 5years after diagnosis.

[0247] In this regard, it may be worth noting that the naturaluteroglobin gene is dependent upon hormones for expression, and hormonalablation may decrease expression of the endogenous uteroglobin gene,both in tumor cells and in the normal tissue surrounding the tumor. Auteroglobin-deficient state could render the patient more susceptible tosuccessful metastasis. Continuous supplementation with uteroglobin-baseddrugs may be used to prevent or reverse this potentiallymetastasis-permissive state from developing in hormonal therapytreatment modalities.

[0248] Chemotherapy

[0249] Chemotherapy has been more successful with some cancers than withothers. It is likely that the combination of chemotherapy with therapiesof the present invention in some cases will be synergistic.

[0250] Examples of chemotherapeutic agents include the compounds of theTable below.

[0251] Immunotherapy

[0252] The present invention also can be used in conjunction withimmunotherapies. Not only may the methods and compositions hereindisclosed be used with the increasing variety of immunological reagentsnow being tested or used to treat cancer, but it also may be used withthose that come into practice in the future. The present invention thusmay be used with immunotherapies based on polyclonal or monoclonalantibody-derived reagents, for instance. Monoclonal antibody-basedreagents are among those most highly preferred in this regard. Suchreagents are well known and are described in, for instance, RitterMONOCLONAL ANTIBODIES - PRODUCTION, ENGINEERING AND CLINICALAPPLICATIONS, Ritter et al., Eds., Cambridge University Press,Cambridge, UK (1995), which is incorporated by reference herein in itsentirety. Radiolabelled monoclonal antibodies for cancer therapy, inparticular, also are well known and are described in, for instance,CANCER THERAPY WITH RADIOLABELLED ANTIBODIES, D. M. Goldenberg, Ed., CRCPress, Boca Raton, Fla. (1995), which is incorporated by referenceherein in its entirety.

[0253] Cryotherapy

[0254] Cryotherapy recently has been applied to the treatment of somecancers. Methods and compositions of the present invention also can beused in conjunction with an effective therapy of this type.

[0255] Compositions Comprising Aeveral Active Agents

[0256] Pharmaceutical compositions of matter useful for inhibitingcancer metastases may contain, in addition to the aforementionedcompounds, an additional therapeutic agent. Such agents may bechemotherapeutic agents, ablation or other therapeutic hormones,antineoplastic agents, monoclonal antibodies useful against cancers andangiogenesis inhibitors. Among hormones which may be used in combinationwith the present invention diethylstilbestrol (DES), leuprolide,flutamide, cyproterone acetate, ketoconazole and amino glutethimide arepreferred.

[0257] Among antineoplastic and anticancer agents that may be used incombination with the invention 5-fluorouracil, vinblastine sulfate,estramustine phosphate, suramin and strontium-89 are preferred.

[0258] Among the monoclonal antibodies that may be used in combinationwith the invention CYT356 is preferred.

[0259] The following table provides known median dosages for selectedcancer agents which may be useful in combination with the compounds andcompositions of the present invention. It should be noted that specificdose levels for the chemotherapeutic agents below will depend uponsimilar dosing considerations as those listed in the DOSAGE sectionpresented herein. NAME OF CHEMOTHERAPEUTIC AGENT MEDIAN DOSAGEAsparaginase 10,000 units Bleomycin Sulfate 15 units Carboplatin 50-450mg. Carmustine 100 mg. Cisplatin 10-50 mg. Cladribine 10 mg.Cyclophosphamide 100 mg.-2 gm. (lyophilized) Cyclophosphamide (non- 100mg.-2 gm. lyophilized) Cytarabine (lyophilized 100 mg.-2 gm. powder)Dacarbazine 100 mg.-200 mg. Dactinomycin 0.5 mg. Daunorubicin 20 mg.Diethylstilbestrol 250 mg. Doxorubicin 10-150 mg. Etidronate 300 mg.Etoposide 100 mg. Floxuridine 500 mg. Fludarabine Phosphate 50 mg.Fluorouracil 500 mg.-5 gm. Goserelin 3.6 mg. Granisetron Hydrochloride 1mg. Idarubicin 5-10 mg. Ifosfamide 1-3 gm. Leucovorin Calcium 50-350 mg.Leuprolide 3.75-7.5 mg. Mechlorethamine 10 mg. Medroxyprogesterone 1 gm.Melphalan 50 gm. Methotrexate 20 mg.-1 gm. Mitomycin 5-40 mg.Mitoxantrone 20-30 mg. Ondansetron Hydrochloride 40 mg. Paclitaxel 30mg. Pamidronate Disodium 30-*90 mg. Pegaspargase 750 units Plicamycin2,500 mcgm. Streptozocin 1 gm. Thiotepa 15 mg. Teniposide 50 mg.Vinblastine 10 mg. Vincristine 1-5 mg. Aldesleukin 22 million unitsEpoetin Alfa 2,000-10,000 units Filgrastim 300-480 mcgm. Immune Globulin500 mg.-10 gm. Interferon Alpha-2a 3-36 million units InterferonAlpha-2b 3-50 million units Levamisole 50 mg. Octreotide 1,000-5,000mcgm. Sargramostim 250-500 mcgm.

[0260] The present invention is further described by reference to thefollowing, illustrative examples.

Example 1 Preparation of Human Uteroglobin by Gene Expression

[0261] Human uteroglobin was purified from E. coli cells expressing afull-length cDNA. The methods for obtaining the cDNA, constructing itinto a vector for expression in host, expressing the construct andpurifying the protein all involve art routine techniques. Such methodsare described specifically with regard to human uteroglobin in Singh etal., BBA 950: 329-337 (1988), Mantile et al., J. Biol Chem. 268:20343-20351 (1993) and Miele et al., J. Biol. Chem. 265: 6427-6435(1990), which are incorporated by reference herein in their entirety.

[0262] Briefly, in the present illustrative example, which followed thetechniques set out in the foregoing references, a clone containing afull-length cDNA encoding human uteroglobin in the well known vectorpGEM4Z was digested with PstI. (pGEM4Z may be obtained from Promega,Inc. Many other equally suitable vectors also are availablecommercially.) The digestion freed a 340-base pair fragment containingall of the cDNA and 53 nucleotides of the pGEM4Z polylinker. Thefragment was purified by preparative gel electrophoresis in low meltingtemperature agarose. The purified fragment was ligated into the Pstlsite of the expression vector pLD101, downstream of an induciblepromoter. The ligation and subsequent cloning produced the plasmidpGEL101. This construct was introduced into E. coli strain BL21(DE3)cells for expression of uteroglobin protein.

[0263] For expression, bacteria were cultured under routine conditionsfor E. coli growth, and then induced for uteroglobin expression bymaking the media 0.45 mM in IPTG (isopropyl-1-thio-D-galactopyranoside).After appropriate further incubation to accumulate expressed protein,the cells were collected and then lysed. Uteroglobin was purified fromthe lysed cells using standard methods of size exclusion and ionexchange chromatography.

Example 2 Cells for Assays of Metastatic Potential

[0264] The cell lines used in the illustrative embodiments hereindiscussed are well known and readily available. The four cell lines ofthe present examples all were derived from human lung cancer and are ofepithelial cell origin. TSU-Pr1, DU-145 and PC3-M areandrogen-independent. LNCaP is androgen-sensitive.

[0265] DU-145 is described in Stone et al., Int. J. Cancer 21: 274-281(1978), which is incorporated herein by reference in its entirety. Thecell line is available from a variety of sources including, forinstance, the American Type Culture Collection (Rockville, Md.).

[0266] LNCaP is described in Horoszewicz et al., Cancer Res. 43:1809-1818 (1983), which is herein incorporated by reference in itsentirety. This cell line may be obtained from, for instance, theAmerican Type Culture Collection (Rockville, Md.).

[0267] PC3-M is described in Kaighn et al., Invent. Urol. 11:16-23(1976) which is herein incorporated by reference in its entirety.

[0268] TSU-Pr1 is described in Hzumi et al., J. Urology 137:1304-1306(1987) which is incorporated by reference in its entirety.

[0269] Cells of each line were grown and maintained in monolayer culturein α-MEM (minimal essential medium) supplemented with glutamine, 10%fetal bovine serum, penicillin (100 units/ml) and streptomycin (100(g/ml). Cultures were incubated at 37EC in 5% CO₂/95% air. Media wasreplaced every second day.

Example 3 General Assay for Invasiveness of Cells

[0270] 1. Culture

[0271] As described briefly below, invasiveness of cells was assayed bythe methods described in Albini et al., Cancer Research 47: 3239-3245(1987), which is incorporated herein by reference in its entirety.Invasiveness assays and other methods for assessing anti-metastaticaffects, as discussed herein below are described in Leyton et al.,Cancer Research 54:3696-3699 (1994) which is incorporated by referenceherein in its entirety.

[0272] Cells in logarithmic phase were detached from the growth surfaceby brief exposure to 0.25% trypsin, 0.25% EDTA, collected andcentrifuged at 800 X g for 5 min. The pellet was resuspended in SFmedium, counted and seeded into 6 mm dishes, 1.5×10⁶ cells per dish. Thecells then were incubated for 24 hr in media containing zero, 0.01, 0.1and 1.0 μM uteroglobin. After the incubation cells were gently collectedusing a rubber policeman and assayed for invasiveness.

[0273] Fibroblast conditioned media (FCM) served as a chemo-attractantto stimulate invasion. It was prepared by culturing proliferating 3T3cells for 24 hr. in SF medium and then collecting the media, free ofcells. The cell free media thus obtained served as FCM.

[0274] Invasiveness was measured using a polycarbonate membraneprecoated with a reconstituted basement membrane. Well known RBMs aresuitable for this purpose. For example, Albini et al., supra describesRBM of the type employed for these experiments.

[0275] Assays were performed in blind-well Boyden chambers. The lowercompartment of each chamber was filled with 220 μl of FCM, aschemo-attractant, or 220 μl serum-free media, as control for basalinvasiveness. A polycarbonate membrane (12-μm pore size), coated with 25μg/50 μl RBM, was placed over the lower compartment. Tumor cells forassay were added to the upper compartment, 3.0×10⁵ cells per well, andthe chambers then were incubated at 37EC for 6 hr.

[0276] (Reconstituted basement membrane preparations for use inaccordance with the foregoing assay are readily available from numerouscommercial suppliers. One suitable example membrane in this regard is“MATRIGEL” sold by Collaborative Biomedical Products of Bedford, Mass.)

[0277] 2. Quantitation

[0278] Invasive activity was measured by the number of cells thatpenetrated the RBM, as determined by a technique involving crystalviolet staining developed for use with the Boyden chamber. Thetechnique, summarized below, is well known and is described, forinstance, in Frandson et al., Fibrinolysis 6 (Supp4): 71-76 (1992),which is incorporated by reference herein in its entirety.

[0279] The RBM-coated membrane was removed from each chamber at the endof the incubation period. The filters were pinned down to a wax plate,keeping the surface with the invading cells upward. The cells werestained on the filters with 0.5% crystal violet in 25% methanol for 10minutes. Then, the filters were rinsed in distilled water, four times oruntil crystal violet no longer leached into the wash water. After thewash, the surface of each filter that had been in contact with the waxplate was carefully wiped clean with a moist cotton swab, to removenonmigrating cells. The filters then were placed in a 24-well clusterplate and dried overnight.

[0280] Crystal violet in the invading cells on each filter was extractedtwice for 10 minutes into 500 μl aliquots of 0.1 M sodium citrate, 50%ethanol. The amount of crystal violet in the extract was analyzed byabsorbance at 585 nm, using a standard spectrometer.

[0281] 3. Analyses

[0282] Assays were carried out in triplicate for each data point.

[0283] Variance between control and test groups was analyzed forsignificance using the standard repeated measures test for analysis ofvariance. P<0.01 generally was considered indicative of a significanteffect, with exceptions, as noted elsewhere herein.

Example 4 Correlation of Optical Density with Cell Counts, and Assay ofBasal Invasiveness

[0284] To calibrate optical density of the crystal violet extractsagainst the number of migrating cells on filters, cells were counted,seeded at known densities on filters, incubated to allow attachment andwashed. One set of a duplicate set of plates was used for cell counting.The other set was used for staining. For counting, cells were releasedfrom the filters by mild trypsinization and then counted using anautomated cell counter. For staining, after washing, the cells werestained and crystal violet stain then was extracted from the stainedcells, as described in EXAMPLE 3. The optical densities of the extractswere measured by standard spectroscopy. The optical density determinedfor each filter extract was matched with the number of cells attached toits companion filter, as determined by direct counting. These paireddata points served to correlate optical density with cell counts.plotted that displayed the

[0285] In accordance with the foregoing procedure, several densities ofDU-145 cells were seeded into the top chamber of the Boyden jars. Thejars were set up in pairs, and for each pair dye uptake by the cells wasmeasured on one filter and the number of cells was counted on the otherfilter, as described above.

[0286] The results are shown in Table I, which sets out the number ofcells migrating to the lower face of the filters as a function of thenumber of cells seeded in the upper chamber. The number of cellsmigrating to the lower filter surface also is set out as a percentage ofthe total number of cells seeded in the top chamber. The data in theTable are the means of triplicate determinations for each condition, andthe indicated variance is the standard error of the mean.

[0287] At cell seedings greater than 2×10⁵ approximately 22% of cellsinvaded the RBM and migrated through the filter in 6 hr. Seeding highernumbers of cells did not increase invasiveness at 6 hr. (Not shown.)

[0288] The cells were counted using an automated cell counter, such asthe “COULTER MULTISIZER” made by Coulter, Inc. of Hialeah, Fla. By theseexperiments it was determined that an absorbance of 0.1 units of thecrystal violet extract corresponds to approximately 5000 cells thatmigrated through the filter.

[0289] Similar procedures can be applied to calibrate the migrationassay for other cells, to employ the assay to measure the therapeuticactivity of other compositions of the present invention. TABLE 1Relationship between cell invasion and optical absorbance Cells O.D.Cells seeded units^(a) invading Percentage (x 10³) (585 nm) (x 10¹)invasion 100 0.60 ″ 0.1 31.8 ″ 0.2 31.8 ″ 0.2 200 1.50 ″ 0.2 42.0 ″ 2.121.2 ″ 1.0 300 1.20 ″ 0.2 72.3 ″ 2.7 23.0 ″ 0.4

[0290] As shown in FIG. 1, cells of the TSU-Pr1 cell line exhibited thehighest rate of basal invasiveness and the highest FCM-stimulatedinvasiveness. The basal rate for DU-145 cells was 2-fold lower than therate for TSU-Pr1 cells, but the rate of stimulated invasiveness wascomparable for the two cell lines. As shown in FIG. 2, basal andstimulated invasiveness of PC3-M cells both were 2-fold lower than thatobserved for DU-145 cells. (N.B. The scale change in FIG. 2.) LNCaPcells exhibited the lowest basal and the lowest stimulated invasiveness.FCM stimulation increased the invasiveness of this cell line 2-3-fold,also as shown in FIG. 2.

Example 5 Uteroglobin Inhibits Invasiveness of Tumor Cells

[0291] The ability of uteroglobin to inhibit tumor cell invasiveness isillustrated by its effect on cell lines treated with 0.01, 0.1 or 1.0 μMuteroglobin. Each of the four cells lines was incubated for 24 hr. withthese concentrations of uteroglobin. After the incubation period thecells were rinsed and then assayed for invasiveness. Inhibition ofstimulated invasiveness then was quantitatively determined bysubtracting the basal invasiveness from FCM-stimulated invasiveness, foreach treatment group. Finally, the results were calculated as apercentage of the invasiveness of untreated control cells.

[0292] All four cell lines showed a dose-dependent inhibition ofFCM-stimulated invasion, as shown in FIGS. 1 and 2, by the bars forFCM/UG. Notably, uteroglobin did not affect basal invasiveness,indicated by the bars labeled SFM/UG. Table 2 shows the averageinhibition observed in three independent experiments, each of which wasperformed in triplicate. The inhibition of invasiveness by uteroglobinwas found to be significant at the P<0.01 level for all conditions,except for PC3-M and TSU-Pr1 cells treated with 0.01 μM uteroglobin,which were significant at the P<0.05 level. As shown in Table 2, 1.0 μMuteroglobin inhibited invasiveness of DU-145 cells by 60%, PC3-M cellsby 88%, LNCaP cells by 92% and the TSU-Pr1 cells by 59%. TABLE 2Inhibition of tumor cell invasiveness by uteroglobin % Inhibition ofinvasion by uteroglobin Cell lines 0.01 μM 0.1 μM 1.0 μM DU-145 45.4 ±6^(a) 49.9 ± 5^(a) 60.2 ± 11^(a) PC3-M 43.4 ± 15^(b) 82.4 ± 14^(a) 87.9± 11^(a) TSU-Prl 33.5 ± 10^(b) 44.4 ± 11^(a) 58.9 ± 8^(a) LNCaP 71.5 ±10^(a) 81.3 ± 6^(a) 92.3 ± 7^(a)

Example 6 Time Course of Inhibition of Invasiveness by Uteroglobin

[0293] The time course for suppression of invasiveness of DU-145 cellsby uteroglobin was determined over a 24 hr. period. Cells were treated,as described above, for 3, 6, 12, or 24 hr. and then assayed forinvasiveness. The maximum inhibition of invasiveness observed in thecells cultured in the presence of uteroglobin was 74%, at 12 hr. 50%maximum inhibition was observed after 3 hr., and at 24 hr. inhibitionwas 79% of the maximum. FIG. 4 shows these results in graphical form.

Example 7 Uteroglobin Does Not Affect Simple Motility

[0294] Experiments carried out to assess motility per se show thatuteroglobin does not affect normal cell motility, i.e., migration in theabsence of RBM. The results show that uteroglobin specifically inhibitsthe invasion-associated motility of epithelial tumor cells.Invasion-associated motility, which is implicated more specifically inmetastasis than motility per se, involves the synthesis, recruitment, oractivation of several different classes of proteolytic enzymes includingcollagenases, cathepsins, plasminogen activators and a variety ofmetalloproteinases required for degradation of basement membranes andthe ECM. The observation that uteroglobin does not alter cell motility,but inhibits FCM-stimulated invasiveness, indicates that uteroglobinspecifically can inhibit metastatic invasiveness without directlyaltering motility of normal cells. This is an advantageous property forpharmacological intervention where non-specific effects of uteroglobinon normal motility could be disadvantageous.

Example 8 Loss of Uteroglobin Expression is an Indicator of EpithelialCell Cancer Progression

[0295] Immunohistochemical analysis of fresh frozen prostate tissuesfrom surgical specimens were taken from 50 patients.

[0296] Eight slides per patient were analyzed for uteroglobin staining.Slides from 26 patients showed evidence of prostate cancer, while slidesfrom the remaining 24 patients showed only benign glands. The results ofTable 3 and Table 4 demonstrate uteroglobin immunoreactivity in normalprostate, benign prostatic hyperplasia (BPH), and prostatic atrophy; lowbut clearly positive expression in prostatic intraepithelial neoplasia(PIN); positive expression in cancerous glands of Gleason's pattern lessthan or equal to 2 and complete loss of uteroglobin immunoreactivity incancerous glands 3 or greater. In addition, in the one case ofmetastatic prostate cancer that we examined, the prostate cancer cellswithin the lymph node lacked uteroglobin expression.

[0297] Further, Western analysis was performed on prostate tissue fromsix of the patients in this study presenting with cancers of Gleason'sgrade 8 or 9. The UG protein runs at around 10 kDa on an SDSpolyacrylamide gel. This protein was present in normal tissue lysatesbut absent in the lysates taken from cancerous tissue. TABLE 3 SUMMARYOF PATIENT DATA # OF Characteristic Detail PATIENTS N = 50 AGE Range35-76 yrs Median 60.8 yrs RACE African American 22 Caucasian 28 FINALBenign Prostatic Hyperplasia  4 PATHOLOGICAL Bladder Carcinoma  2DIAGNOSIS PIN  5 (The two Prostate CA Gleason's Grade 5  4 patients withProstate CA Gleason's Grade 6 12 bladder Prostate CA Gleason's Grade 720 carcinoma had Prostate CA Gleason's Grade 8  2 a final Prostate CAGleason's Grade 9  7 diagnosis of Prostate CA Gleason's Grade 10  1 BPH.PIN was associated with PC in all five cases; thus N = PC + BPH = 50)SLIDE Benign Prostatic Hyperplasia 42 DIAGNOSIS Prostatic Atrophy  3(Patterns in PIN  2 glands and PC Gleason's Pattern ≧3 20 cells in thePC Gleason's Pattern ≦2  6 field of view Fibromuscular Tissue only  2 atthe time of slide analysis. The numbers here represent n [patients] outof a total of 50 in which these pathologies were observed)

[0298] TABLE 4 SUMMARY OF STAINING FOR UG EXPRESSION Negative LowModerate Strong (0) (+) (++) (+++) BENIGN 19 25 1 PIN  2 CANCER  5  1(≦2) CANCER 20 (≧3)

Example 9 Specificity of Uteroglobin Anti-metastatic Effects

[0299] The specificity of uteroglobin activity was demonstrated inDU-145 cells, using myoglobin, albumin and heat inactivated uteroglobin.Cells were treated for 24 hr with either myoglobin, albumin oruteroglobin that had been inactivated by incubation at 55EC for 45 min.The results, presented in the graph in FIG. 3, show that myoglobin,albumin and heat-inactivated uteroglobin do not effect invasive activityof tumor cells.

Example 10 Uteroglobin Inhibits Arachidonic Acid Release by FCMStimulated Tumor Cells

[0300] The effect of uteroglobin on the release of arachidonic acid(“AA”) by tumor cells was assayed under conditions of basal andstimulated invasiveness. (¹⁴C)AA having a specific activity 58.0mCi/mmol was used to trace the arachidonic acid release. Labeledarachidonic acid of this type can be obtained from several commercialsuppliers, such as Amersham, Inc. of Arlington Heights, Ill.

[0301] Uteroglobin inhibits release of arachidonic acid by DU-145 cellsstimulated by FCM, as shown by the following experiment.

[0302] Intracellular arachidonic acid in DU-145 cells was labeled byincubating the cells in media containing ¹⁴C-labeled arachidonic acid.For this purpose approximately 0.75×10⁵ cells were incubated for 24 hr.at 37EC in 2 ml of α-MEM/SF media containing 1 μCi of (¹⁴C)AA. Afterthis, the cells were washed three times with 20 ml 0.2% bovine serumalbumin to remove free radioactivity.

[0303] The washed, labeled cells were resuspended in 2 ml of α-MEM/SF,FCM or FCM containing 1.0 μM uteroglobin. Cells in each of the threemedia were incubated at 37EC and 50 μl aliquots were removed of themedia were removed from each culture 0.5, 10, 20 and 30 min., and 1, 2,3, 4 and 5 hr. after the beginning of the incubation period.

[0304] Each aliquot was assayed for AA release, which was measured as¹⁴C free in the media, determined by scintillation counting. Standardscintillents and counters were used to quantitate radiation emission by¹⁴C in the samples; e.g., EcoLite Biodegradable scintillant from ICN,Inc.

[0305] Stimulation of AA release by FCM was calculated by subtractingthe amount of (¹⁴C)AA released by cells incubated in α-MEM/SF media(which was very low) from the amount of (¹⁴C)AA released by cellsincubated in FCM media. The effect of uteroglobin on FCM-stimulated AArelease was calculated in the same way, by subtracting the amount of(¹⁴C)AA released by cells cultured in α-MEM/SF from the amount of(¹⁴C)AA released by cells incubated in FCM containing 1 μM uteroglobin.

[0306] As shown in the graph in FIG. 5, arachidonic acid released bycells cultured in FCM media exhibited a biphasic profile. Released AApeaked at 20 min., the peak was followed by a period of reuptake, e.g.60 min. and then there was a period of sustained release to the end ofthe 5 hr. incubation period of this experiment.

[0307] The presence of 1 μM uteroglobin in FCM media reducedFCM-stimulated release of arachidonic by 77% at 20 min. and 86% at 5 hr.

[0308] The dramatic inhibition of release of arachidonic acid byFCM-stimulated tumor cells, together with the foregoing results showingthe inhibitory effect of uteroglobin on invasiveness, show thatuteroglobin affects an early event in the signalling pathway(s) thatcontrol tumor invasiveness.

Example 11

[0309] A patient presents with metastatic adenocarcinoma of the lung.The adenocarcinoma appears not to have metastasized. The adenocarcinomais removed by surgery. Uteroglobin is administered before and aftersurgery at a dose rate that reaches and then maintains a bloodconcentration of uteroglobin of approximately 1 μM. After post-operativerecovery, the patient is maintained at a decreased level of uteroglobinby a regimen of periodic i.m. self-administration. No furtheroccurrences of the adenocarcinoma develop.

Example 12

[0310] A patient presents with metastatic adenocarcinoma of the lung.The adenocarcinoma appears not to have metastasized. The adenocarcinomais removed by surgery. Uteroglobin is administered before and aftersurgery, at a dose rate that reaches and then maintains a bloodconcentration of uteroglobin of approximately 1 μM. After post-operativerecovery, the patient is maintained at a decreased level of uteroglobinby intermittent or continuous administration by subdural pump. Nofurther occurrences of the adenocarcinoma develop.

Example 13

[0311] A patient presents with metastatic adenocarcinoma of the lung.The adenocarcinoma appears not to have metastasized. The adenocarcinomais removed by surgery. Uteroglobin is administered before and aftersurgery, at a dose rate that reaches and then maintains a bloodconcentration of uteroglobin of approximately 1 μM. After post-operativerecovery, the patient is maintained at a decreased level of uteroglobinby intermittent or continuous administration using a transdermal patch.No further occurrences of the adenocarcinoma develop.

Example 14

[0312] A patient presents with metastatic adenocarcinoma of the lung.The adenocarcinoma appears to have metastasized, but surgery still isindicated as an effective treatment modality. Tumor tissue is removed bysurgery. Uteroglobin is administered from the time, approximately, ofthe initial diagnosis and continues after surgery, i.m. and i.v., at adose rate that reaches and then maintains a blood concentration ofuteroglobin above 1 μM. After post-operative recovery, the patient ismaintained at this level of uteroglobin by a regimen of periodic i.m.self-administration. The patient is monitored carefully for intolerableadverse side-effects of high-dose uteroglobin administration. No furthertumors develop.

Example 15

[0313] A patient presents with metastatic adenocarcinoma of the lung.The adenocarcinoma appears to have metastasized, but surgery still isindicated as an effective treatment modality. Tumor tissue is removed bysurgery. Uteroglobin is administered from the time, approximately, ofthe initial diagnosis and continues after surgery, i.m. and i.v., at adose rate that reaches and then maintains a blood concentration ofuteroglobin above 1 μM. After post-operative recovery, the patient ismaintained at this level of uteroglobin by a regimen of periodic i.m.self-administration. The patient is monitored carefully for intolerableadverse side-effects of high-dose uteroglobin administration. Althoughsome of the original, small tumorous masses are detected after surgery,they do not grow in size.

Example 16

[0314] A patient presents with metastatic adenocarcinoma of the lung.The adenocarcinoma appears to have metastasized, but surgery still isindicated as an effective treatment modality. Tumor tissue is removed bysurgery. Uteroglobin is administered from the time, approximately, ofthe initial diagnosis and continues after surgery, i.m. and i.v., at adose rate that reaches and then maintains a blood concentration ofuteroglobin above 1 μM. After post-operative recovery, the patient ismaintained at this level of uteroglobin by a regimen of periodic i.m.self-administration. The patient is monitored carefully for intolerableadverse side-effects of high-dose uteroglobin administration. Tumorousmasses are detected after surgery, but their growth is slowed.

Example 17

[0315] A patient presents with a tumor of the lung, of epithelial cellorigin. The tumor, which appears to be of a metastatic type, appears notto have metastasized. The tumor is removed by surgery. Uteroglobin isadministered after surgery, i.m. and i.v., at a dose rate that reachesand then maintains a blood concentration of uteroglobin of approximately1 μM. After post-operative recovery, the patient is maintained at adecreased level of uteroglobin by a regimen of periodic i.m.self-administration. No further occurrences of the tumor develop.

Example 18

[0316] A patient presents with a lung tumor of epithelial cell origin.The tumor has metastasized. Numerous secondary tumors are detected.Insofar as possible, tumor tissue is removed by surgery. Surgicalintervention is aggressive. Uteroglobin is administered from the time,approximately, of the initial diagnosis and continues after surgery,i.m. and i.v., at a dose rate that reaches and then maintains a bloodconcentration of uteroglobin above 1 μM. After post-operative recovery,the patient is maintained at this level of uteroglobin by a regimen ofperiodic i.m. self-administration. The patient is monitored carefullyfor intolerable adverse side-effects of high-dose uteroglobinadministration. No further tumors develop in the remaining lung orelsewhere in the body.

Example 19 Uteroglobin is not Expressed or Secreted by Lung CarcinomaCells

[0317] A 76 year old patient would undergo surgery to remove a cancerousportion of the lung. Postsurgical definitive pathologic diagnosis showedmoderately to poorly differentiated adenocarcinoma. Perineural andlymphatic invasion was noted.

[0318] With informed consent and in accordance with approved procedures,lung tissue is obtained from the diseased lung gland after removal, forevaluation of uteroglobin expression. Tissue fragments are frozen inliquid nitrogen. Individual slices of frozen tissue are sectioned bycryostat and mounted on silanated microscope slides.

[0319] Samples are fixed with 4% formalin for 3 minutes at roomtemperature and washed for 10 min in phosphate buffered saline (PBS) pH8.0. Nonspecific reactivity is blocked by incubating samples with rabbitserum (1:100 dilution) for 30 min. The samples are then exposed to a1:1,000 dilution of goat anti-human uteroglobin primary antibodyovernight at 4 degrees C. Control samples are exposed to a 1:100dilution of goat serum.

[0320] All samples are then exposed to biotinylated rabbit anti-goatantibody (1:10,000) for 30 min and then washed with PBS for 10 min.Streptavidin complex is added for 15 min and the samples washed againwith PBS for 10 min. DAB is reacted with the samples for 2 min followedby standard staining of the tissue with hematoxylin-eosin.

[0321] Slides are analyzed by certified pathologists, who are notinformed of the identity of any slides and who carry out their analysesindependently.

[0322] In each case, the normal lung tissue is expected to stainstrongly positive for uteroglobin in epithelial cells, especially at theintracellular luminal surface. Stromal cells are negative. Areas ofhyperplasia diagnosed independently by both pathologists stainpositively. In contrast, tumorous epithelial cells exhibit little or nostaining for uteroglobin, demonstrating that the tumor cells had lostthe ability to synthesize and secrete this protein.

Example 20 Uteroglobin mRNA is not Detected or is Aberrantly Processedin Cells Derived from Metastases of Human Lung Tumors

[0323] RNA was isolated from normal lung tissue and from the DU-145,PC-3 and TSU-PR1 cell lines derived from metastatic human lung tumors.The RNA was subjected to Northern blotting according to routine andstandard procedures, using a radiolabelled uteroglobin cDNA probe.Autoradiographic analysis showed that the normal tissue expresses anabundant normally processed 600 base pair mRNA uteroglobin transcript.In contrast the metastatic tumor cells either did not detectably expressthe uteroglobin transcript (DU-145 and PC-3) or expressed a grosslyaberrant transcript (TSU-PR1).

Example 21

[0324] A patient presents with bronchial obstruction and afterradiographic exam a biopsy of the lung is taken. The initial presurgicalreport suggests a low-grade localized tumor. A portion of the biopsysample is analyzed immunohistochemically for uteroglobin expressionwhich is found to be present in the normal tissue but absent in thetumor cells. The diagnosis, prognosis, and plan for therapy isappropriately altered to reflect the high probability, based on lack ofuteroglobin expression, that the tumor is actually of higher grade thaninitially diagnosed and probably invasive and metastatic. Uteroglobintherapy is immediately begun.

Example 22

[0325] After surgery to remove tumorous from the lung, a patientpresents with high grade metastatic lung adenocarcinoma that has becomerefractory to hormonal therapy. The patient refuses chemotherapy basedon its dismal efficacy against lung cancer and its devastating sideeffects. In situ hybridization analysis of a tumor biopsy reveals thatthe uteroglobin gene is not being expressed in the tumor cells. Furtheranalysis of uteroglobin gene structure by SSCP and RFLP indicate thethat the gene is mutated and dysfunctional. The patient chooses tobecome a candidate for gene therapy. The patient is injected with anadenovirus-based plasmid expression vector containing the uteroglobingene linked to the promotor of the PSA gene which is specificallyexpressed in lung cells. The vector is encapsulated in liposomes whichhave anti-PSA antibody fixed on the surface. The antibody-liposomecomplex binds specifically to cells secreting PSA which presumably areonly the metastatic tumor cells. The liposomes are ingested by the cellsand release the plasmids which incorporate into the cells' genomic DNAand begin expressing uteroglobin. The transfected cells expressinguteroglobin reverse their invasive phenotype, thereby ceasing furthermetastasis and are gradually destroyed by the bodies natural defenses.The metastatic tumors regress and the patient's life is prolonged.

[0326] The invention being thus described, it will be obvious that thesame may be varied in many ways. Such variations are not to be regardedas a departure from the spirit and scope of the invention and all suchmodification are intended to be included within the scope of thefollowing claims.

What is claimed is:
 1. A method for treatment of lung cancer, whichcomprises administering a therapeutically effective amount of aninhibitor of phospholipase A₂ to an animal.
 2. The method of claim 1,wherein the inhibitor of phospholipase A₂ is uteroglobin, a mutein ofuteroglobin, a peptide analog of uteroglobin or a mimetic ofuteroglobin.
 3. The method of claim 1, wherein the inhibitor ofphospholipase A₂ is administered in combination with an additional agentselected from the group consisting of radiotherapeutic agents, hormonaltherapy agents, immunotherapeutic agents, chemotherapeutic agents,cryotherapeutic agents and gene therapy agents.
 4. The method of claim1, wherein the inhibitor of phospholipase A₂ is locally administered tolung epithelial tissue.
 5. A pharmaceutical composition for treatment oflung cancer in an animal, which comprises a therapeutically effectiveamount of an inhibitor of phospholipase A₂ and a pharmaceuticallyacceptable carrier.
 6. The pharmaceutical composition of claim 5,wherein the inhibitor of phospholipase A₂ is uteroglobin, a mutein ofuteroglobin, a peptide analog of uteroglobin or a mimetic ofuteroglobin.
 7. The pharmaceutical composition of claim 5, whichcomprises an additional agent selected from the group consisting ofradiotherapeutic agents, hormonal therapy agents, immunotherapeuticagents, chemotherapeutic agents, cryotherapeutic agents and gene therapyagents.
 8. The pharmaceutical composition of claim 5, wherein theinhibitor of phospholipase A₂ is formulated for local administration tolung epithelial tissue.
 9. A method for inhibiting metastasis of lungcancer cells, which comprises administering a therapeutically effectiveamount of an inhibitor of phospholipase A₂ to an animal.
 10. The methodof claim 9, wherein the inhibitor of phospholipase A₂ is uteroglobin, amutein of uteroglobin, a peptide analog of uteroglobin or a mimetic ofuteroglobin.
 11. The method of claim 9, wherein the inhibitor ofphospholipase A₂ is administered in combination with an additional agentselected from the group consisting of radiotherapeutic agents, hormonaltherapy agents, immunotherapeutic agents, chemotherapeutic agents,cryotherapeutic agents and gene therapy agents.
 12. The method of claim9, wherein the inhibitor of phospholipase A₂ is locally administered tolung epithelial tissue.
 13. A pharmaceutical composition for inhibitingmetastasis of lung cancer cells in an animal, which comprises atherapeutically effective amount of an inhibitor of phospholipase A₂ anda pharmaceutically acceptable carrier.
 14. The pharmaceuticalcomposition of claim 13, wherein the inhibitor of phospholipase A₂ isuteroglobin, a mutein of uteroglobin, a peptide analog of uteroglobin ora mimetic of uteroglobin.
 15. The pharmaceutical composition of claim13, which comprises an additional agent selected from the groupconsisting of radiotherapeutic agents, hormonal therapy agents,immunotherapeutic agents, chemotherapeutic agents, cryotherapeuticagents and gene therapy agents.
 16. The pharmaceutical composition ofclaim 13, wherein the inhibitor of phospholipase A₂ is formulated forlocal administration to lung epithelial tissue.
 17. A method forinhibiting invasion of metastatic lung cancer cells, which comprisesadministering a therapeutically effective amount of an inhibitor ofphospholipase A₂ to an animal.
 18. The method of claim 17, wherein theinhibitor of phospholipase A₂ is uteroglobin, a mutein of uteroglobin, apeptide analog of uteroglobin or a mimetic of uteroglobin.
 19. Themethod of claim 17, wherein the inhibitor of phospholipase A₂ isadministered in combination with an additional agent selected from thegroup consisting of radiotherapeutic agents, hormonal therapy agents,immunotherapeutic agents, chemotherapeutic agents, cryotherapeuticagents and gene therapy agents.
 20. The method of claim 17, wherein theinhibitor of phospholipase A₂ is locally administered to lung epithelialtissue.
 21. A pharmaceutical composition for inhibiting invasion ofmetastatic lung cancer cells in an animal, which comprises atherapeutically effective amount of an inhibitor of phospholipase A₂ anda pharmaceutically acceptable carrier.
 22. The pharmaceuticalcomposition of claim 21, wherein the inhibitor of phospholipase A₂ isuteroglobin, a mutein of uteroglobin, a peptide analog of uteroglobin ora mimetic of uteroglobin.
 23. The pharmaceutical composition of claim21, which comprises an additional agent selected from the groupconsisting of radiotherapeutic agents, hormonal therapy agents,immunotherapeutic agents, chemotherapeutic agents, cryotherapeuticagents and gene therapy agents.
 24. The pharmaceutical composition ofclaim 21, wherein the inhibitor of phospholipase A₂ is formulated forlocal administration to lung epithelial tissue.
 25. A method fordetecting or identifying metastatic lung cancer, which comprises:comparing an amount of an inhibitor of phospholipase A₂ in a sample oflung epithelial tissue to at least one reference which correlates to anamount of the inhibitor of phospholipase A₂ in a sample of normal lungepithelial tissue, or in a sample of metastatic lung cancer, wherebydifferential amounts of the inhibitor of phospholipase A₂ between thesample of lung epithelial tissue and the reference which correlates tonormal lung epithelial tissue, or to metastatic lung cancer, detects oridentifies metastatic lung cancer.
 26. The method of claim 25, whereinthe inhibitor of phospholipase A₂ is uteroglobin, a mutein ofuteroglobin, a peptide analog of uteroglobin or a mimetic ofuteroglobin.
 27. The method of claim 26, wherein the amount ofuteroglobin is determined by the amount of uteroglobin mRNA or DNA inthe sample.
 28. The method of claim 27, wherein the amount ofuteroglobin mRNA is determined by in situ hybridization.
 29. The methodof claim 25, wherein the inhibitor is detected using ELISA immunoassay,radioimmunoassay, chemiluminescence immunoassay, fluorescenceimmunoassay, cell sorting assay, fluorescence activated cell sortingassay, Western blotting techniques, immunoprecipitation assay,colorimetric or densitometric assay, enzymatic assay, and immunostainingassay.
 30. The method of claim 25, wherein the reference whichcorrelates to an amount of the inhibitor of phospholipase A₂ in a sampleof normal lung epithelial tissue, or in a sample of metastatic lungcancer includes a series of standards which indicate an amount of theinhibitor of phospholipase A₂ and provides a gradient from lower amountsto higher amounts of the inhibitor, wherein higher amounts of inhibitorindicates normal lung epithelial cells and lower amounts of inhibitorindicates metastatic lung cancer.
 31. The method of claim 25, whereinthe sample of lung epithelial tissue is selected from the groupconsisting of lung biopsy, sputum, bronchial fluid, bronchial aspirate,and pleural fluid.
 32. A method for detecting or identifying metastaticlung cancer, which comprises: assaying for inhibitor of phospholipase A₂in a sample of lung epithelial tissue; and comparing the amount of theinhibitor of phospholipase A₂ in the sample of lung epithelial tissue toa reference which correlates to an amount of the inhibitor ofphospholipase A₂ in a sample of normal lung epithelial tissue, or in asample of metastatic lung cancer, whereby differential amounts of theinhibitor of phospholipase A₂ between the sample of lung epithelialtissue and the reference which correlates to normal lung epithelialtissue, or to metastatic lung cancer detects or identifies metastaticlung cancer.
 33. The method of claim 32, wherein the inhibitor ofphospholipase A₂ is uteroglobin, a mutein of uteroglobin, a peptideanalog of uteroglobin or a mimetic of uteroglobin.
 34. The method ofclaim 33, wherein the amount of uteroglobin is determined by the amountof uteroglobin mRNA or DNA in the sample.
 35. The method of claim 34,wherein the amount of uteroglobin mRNA is determined by in situhybridization.
 36. The method of claim 32, wherein the inhibitor isdetected using ELISA immunoassay, radioimmunoassay, chemiluminescenceimmunoassay, fluorescence immunoassay, cell sorting assay, fluorescenceactivated cell sorting assay, Western blotting techniques,immunoprecipitation assay, colorimetric or densitometric assay,enzymatic assay, and immunostaining assay.
 37. The method of claim 32,wherein the reference which correlates to an amount of the inhibitor ofphospholipase A₂ in a sample of normal lung epithelial tissue, or in asample of metastatic lung cancer includes a series of standards whichindicate an amount of the inhibitor of phospholipase A₂ and provides agradient from lower amounts to higher amounts of the inhibitor, whereinhigher amounts of inhibitor indicates normal lung epithelial cells andlower amounts of inhibitor indicates metastatic lung cancer.
 38. Themethod of claim 32, wherein the sample of lung epithelial tissue isselected from the group consisting of lung biopsy, sputum, bronchialfluid, bronchial aspirate, and pleural fluid.
 39. A method for detectingor identifying a pathological condition of lung epithelial tissue, whichcomprises: assaying for inhibitor of phospholipase A₂ in a sample oflung epithelial tissue; and comparing the amount of the inhibitor ofphospholipase A₂ in the sample of lung epithelial tissue to a referencewhich correlates to an amount of the inhibitor of phospholipase A₂ in asample of normal lung epithelial tissue, or in a sample of metastaticlung cancer, whereby differential amounts of the inhibitor ofphospholipase A₂ between the sample of lung epithelial tissue and thereference which correlates to normal lung epithelial tissue, or tometastatic lung cancer detects or identifies a pathological condition oflung tissue.
 40. The method of claim 39, wherein the inhibitor ofphospholipase A₂ is uteroglobin, a mutein of uteroglobin, a peptideanalog of uteroglobin or a mimetic of uteroglobin.
 41. The method ofclaim 40, wherein the amount of uteroglobin is determined by the amountof uteroglobin mRNA or DNA in the sample.
 42. The method of claim 41,wherein the amount of uteroglobin mRNA is determined by in situhybridization.
 43. The method of claim 39, wherein the inhibitor isdetected using ELISA immunoassay, radioimmunoassay, chemiluminescenceimmunoassay, fluorescence immunoassay, cell sorting assay, fluorescenceactivated cell sorting assay, Western blotting techniques,immunoprecipitation assay, colorimetric or densitometric assay,enzymatic assay, and immunostaining assay.
 44. The method of claim 39,wherein the reference which correlates to an amount of the inhibitor ofphospholipase A₂ in a sample of normal lung epithelial tissue, or in asample of metastatic lung cancer includes a series of standards whichindicate an amount of the inhibitor of phospholipase A₂ and provides agradient from lower amounts to higher amounts of the inhibitor, whereinhigher amounts of inhibitor indicates normal lung epithelial cells andlower amounts of inhibitor indicates metastatic lung cancer.
 45. Themethod of claim 39, wherein the sample of lung epithelial tissue isselected from the group consisting of lung biopsy, sputum, bronchialfluid, bronchial aspirate, and pleural fluid.
 46. A method for detectingor identifying an inhibitor of phospholipase A₂ in a sample of lungepithelial tissue, which comprises: assaying for an inhibitor ofphospholipase A₂ in a sample of lung epithelial.
 47. The method of claim46, wherein the inhibitor of phospholipase A₂ is uteroglobin, a muteinof uteroglobin, a peptide analog of uteroglobin or a mimetic ofuteroglobin.
 48. The method of claim 47, wherein the amount ofuteroglobin is determined by the amount of uteroglobin mRNA or DNA inthe sample.
 49. The method of claim 48, wherein the amount ofuteroglobin mRNA is determined by in situ hybridization.
 50. The methodof claim 46, wherein the inhibitor is detected using ELISA immunoassay,radioimmunoassay, chemiluminescence immunoassay, fluorescenceimmunoassay, cell sorting assay, fluorescence activated cell sortingassay, Western blotting techniques, immunoprecipitation assay,colorimetric or densitometric assay, enzymatic assay, and immunostainingassay.
 51. The method of claim 46, wherein the sample of lung epithelialtissue is selected from the group consisting of lung biopsy, sputum,bronchial fluid, bronchial aspirate, and pleural fluid.
 52. A kit fortreatment of lung cancer, which comprises a therapeutically effectiveamount of an inhibitor of phospholipase A₂ in a pharmaceuticallyacceptable carrier and a device for delivery of the inhibitor to thelung cancer, wherein the inhibitor, the carrier and the device arepackaged in a container.
 53. The kit of claim 52, wherein the inhibitorof phospholipase A₂ is uteroglobin, a mutein of uteroglobin, a peptideanalog of uteroglobin or a mimetic of uteroglobin.
 54. The kit of claim52, further which comprises an additional agent selected from the groupconsisting of radiotherapeutic agents, hormonal therapy agents,immunotherapeutic agents, chemotherapeutic agents, cryotherapeuticagents and gene therapy agents.
 55. A kit for inhibiting metastasis oflung cancer, which comprises a therapeutically effective amount of aninhibitor of phospholipase A₂ in a pharmaceutically acceptable carrierand a device for delivery of the inhibitor to the lung cancer, whereinthe inhibitor, the carrier and the device are packaged in a container.56. The kit of claim 55, wherein the inhibitor of phospholipase A₂ isuteroglobin, a mutein of uteroglobin, a peptide analog of uteroglobin ora mimetic of uteroglobin.
 57. The kit of claim 55, further whichcomprises an additional agent selected from the group consisting ofradiotherapeutic agents, hormonal therapy agents, immunotherapeuticagents, chemotherapeutic agents, cryotherapeutic agents and gene therapyagents.
 58. A kit for inhibiting invasion of lung cancer, whichcomprises a therapeutically effective amount of an inhibitor ofphospholipase A₂ in a pharmaceutically acceptable carrier and a devicefor delivery of the inhibitor to the lung cancer, wherein the inhibitor,the carrier and the device are packaged in a container.
 59. The kit ofclaim 58, wherein the inhibitor of phospholipase A₂ is uteroglobin, amutein of uteroglobin, a peptide analog of uteroglobin or a mimetic ofuteroglobin.
 60. The kit of claim 58, further which comprises anadditional agent selected from the group consisting of radiotherapeuticagents, hormonal therapy agents, immunotherapeutic agents,chemotherapeutic agents, cryotherapeutic agents and gene therapy agents.61. A kit for detecting or identifying metastatic lung cancer, whichcomprises: means for collecting a sample of lung epithelial cells; meansfor detecting an inhibitor of phospholipase A₂ in the sample of lungepithelial cells; and at least one reference which correlates to anamount of the inhibitor of phospholipase A₂ in normal lung epithelialcells or in metastatic lung cancer cells, wherein differential amountsof the inhibitor of phospholipase A₂ between the sample of lungepithelial cells and the reference which correlates to normal lungepithelial cells or metastatic lung cancer cells detects or identifiesmetastatic lung cancer.
 62. The kit of claim 61, wherein the inhibitorof phospholipase A₂ is uteroglobin, a mutein of uteroglobin, a peptideanalog of uteroglobin or a mimetic of uteroglobin.
 63. The kit of claim62, wherein the amount of uteroglobin is detected by the amount ofuteroglobin mRNA or DNA in the sample.
 64. The kit of claim 63, whereinthe amount of uteroglobin mRNA is detected by in situ hybridization. 65.The kit of claim 61, wherein the inhibitor is detected using ELISAimmunoassay, radioimmunoassay, chemiluminescence immunoassay,fluorescence immunoassay, cell sorting assay, fluorescence activatedcell sorting assay, Western blotting techniques, immunoprecipitationassay, colorimetric or densitometric assay, enzymatic assay, andimmunostaining assay.
 66. The kit of claim 61, wherein the referencewhich correlates to the amount of inhibitor of phospholipase A₂ innormal lung epithelial cells or metastatic lung cancer cells, includes aseries of standards which indicate an amount of the inhibitor ofphospholipase A₂ and provides a gradient from lower amounts to higheramounts of the inhibitor, wherein higher amounts of inhibitor indicatesnormal lung epithelial cells and lower amounts of inhibitor indicatesmetastatic lung cancer.
 67. The kit of claim 61, wherein the sample oflung epithelial tissue is selected from the group consisting of lungbiopsy, sputum, bronchial fluid, bronchial aspirate, and pleural fluid.68. A kit for detecting or identifying a pathological condition of lungepithelial cells, which comprises: means for collecting a sample of lungepithelial cells; means for detecting an inhibitor of phospholipase A₂in the sample of lung epithelial cells; and at least one reference whichcorrelates to an amount of the inhibitor of phospholipase A₂ in normallung epithelial cells or in metastatic lung cancer cells, whereindifferential amounts of the inhibitor of phospholipase A₂ between thesample of lung epithelial cells and the reference which correlates tonormal lung epithelial cells or metastatic lung cancer cells detects oridentifies a pathological condition of the lung epithelial cells. 69.The kit of claim 68, wherein the inhibitor of phospholipase A₂ isuteroglobin, a mutein of uteroglobin, a peptide analog of uteroglobin ora mimetic of uteroglobin.
 70. The kit of claim 69, wherein the amount ofuteroglobin is detected by the amount of uteroglobin mRNA or DNA in thesample.
 71. The kit of claim 70, wherein the amount of uteroglobin mRNAis detected by in situ hybridization.
 72. The kit of claim 68, whereinthe inhibitor is detected using ELISA immunoassay, radioimmunoassay,chemiluminescence immunoassay, fluorescence immunoassay, cell sortingassay, fluorescence activated cell sorting assay, Western blottingtechniques, immunoprecipitation assay, colorimetric or densitometricassay, enzymatic assay, and immunostaining assay.
 73. The kit of claim68, wherein the reference which correlates to the amount of inhibitor ofphospholipase A₂ in normal lung epithelial cells or metastatic lungcancer cells, includes a series of standards which indicate an amount ofthe inhibitor of phospholipase A₂ and provides a gradient from loweramounts to higher amounts of the inhibitor, wherein higher amounts ofinhibitor indicates normal lung epithelial cells and lower amounts ofinhibitor indicates metastatic lung cancer.
 74. The kit of claim 68,wherein the sample of lung epithelial tissue is selected from the groupconsisting of lung biopsy, sputum, bronchial fluid, bronchial aspirate,and pleural fluid.
 75. A kit for detecting or identifying an inhibitorof phospholipase A₂ in a sample of lung epithelial cells, whichcomprises: means for collecting a sample of lung epithelial cells; andmeans for detecting an inhibitor of phospholipase A₂ in the sample oflung epithelial cells.
 76. The kit of claim 75, wherein the inhibitor ofphospholipase A₂ is uteroglobin, a mutein of uteroglobin, a peptideanalog of uteroglobin or a mimetic of uteroglobin.
 77. The kit of claim76, wherein the amount of uteroglobin is detected by the amount ofuteroglobin mRNA or DNA in the sample.
 78. The kit of claim 77, whereinthe amount of uteroglobin mRNA is detected by in situ hybridization. 79.The kit of claim 75, wherein the inhibitor is detected using ELISAimmunoassay, radioimmunoassay, chemiluminescence immunoassay,fluorescence immunoassay, cell sorting assay, fluorescence activatedcell sorting assay, Western blotting techniques, immunoprecipitationassay, colorimetric or densitometric assay, enzymatic assay, andimmunostaining assay.
 80. The kit of claim 75, wherein the sample oflung epithelial tissue is selected from the group consisting of lungbiopsy, sputum, bronchial fluid, bronchial aspirate, and pleural fluid.81. A method for detecting or identifying metastatic lung cancer in ananimal, which comprises: assaying for an aberrant uteroglobin protein ina sample of lung epithelial cell tissue, wherein a positive indicationfor aberrant uteroglobin detects metastatic lung cancer.
 82. The methodof claim 81, wherein the sample of lung epithelial tissue is selectedfrom the group consisting of lung biopsy, sputum, bronchial fluid. 83.The method of claim 81, wherein the aberrant uteroglobin is detectedusing ELISA immunoassay, radioimmunoassay, chemiluminescenceimmunoassay, fluorescence immunoassay, cell sorting assay, fluorescenceactivated cell sorting assay, Western blotting techniques,immunoprecipitation assay, colorimetric or densitometric assay,enzymatic assay, and immunostaining assay.
 84. A kit for detecting oridentifying metastatic lung cancer in an animal, which comprises: meansfor detecting an aberrant uteroglobin in a sample of lung epithelialcells, wherein a positive indication of aberrant uteroglobin detects oridentifies metastatic lung cancer.
 85. The kit of claim 84, wherein thesample of lung epithelial tissue is selected from the group consistingof lung biopsy, sputum, bronchial fluid.
 86. The kit of claim 84,wherein the aberrant uteroglobin is detected using ELISA immunoassay,radioimmunoassay, chemiluminescence immunoassay, fluorescenceimmunoassay, cell sorting assay, fluorescence activated cell sortingassay, Western blotting techniques, immunoprecipitation assay,colorimetric or densitometric assay, enzymatic assay, and immunostainingassay.
 87. The kit of claim 84, further comprising at least onereference or control which correlates to aberrant uteroglobin or normaluteroglobin.